Novel methods for displaying (poly) pepti des/proteins on bacteriophage particles via disulfide bonds

ABSTRACT

The present invention relates to methods for displaying (poly)peptides/proteins on the surface of bacteriophage particles by attaching the (poly)peptide/proteins via disulfide bonds.

The present invention relates to methods for displaying (poly)peptides/proteins on the surface of bacteriophage particles by attaching the (poly)peptide/proteins via disulfide bonds.

[0001] This application is based upon, and claims priority to, European patent applications EP 99 11 4072.4 and EP 00 10 3551.8 which are incorporated herein by reference in their entirety. A number of documents are cited throughout this specification. The disclosure content of these documents is herewith incorporated by reference in their entirety.

[0002] Smith first demonstrated in 1985 that filamentous phage tolerate foreign protein fragments inserted in their gene III protein (pIII), and could show that the protein fragments are presented on the phage surface (Smith, 1985). Ladner extended that concept to the screening of repertoires of (poly)peptides and/or proteins displayed on the surface of phage (WO 88/06630; WO 90/02809) and, since then, phage display has experienced a dramatic progress and resulted in substantial achievements. Various formats have been developed to construct and screen (poly)peptide/protein phage-display libraries, and a large number of review articles and monographs cover and summarise these developments (e.g., Kay et al., 1996; Dunn, 1996; McGregor, 1996). Most often, filamentous phage-based systems have been used. Initially proposed as display of single-chain Fv (scFv) fragments (WO 88/06630; see additionally WO 92/01047), the method has rapidly been expanded to the display of bovine pancreatic trypsin inhibitor (BPTI) (WO 90/02809), peptide libraries (WO 91/19818), human growth hormone (WO 92/09690), and of various other proteins including the display of multimeric proteins such as Fab fragments (WO 91/17271; WO 92/01047). To anchor the peptide or protein to the filamentous bacteriophage surface, mostly genetic fusions to phage coat proteins are employed. Preferred are fusions to gene III protein (Parmley & Smith, 1988) or fragments thereof (Bass et al., 1990), and gene VIII protein (Greenwood et al., 1991). In one case, gene VI has been used (Jespers et al., 1995), and recently, a combination of gene VII and gene IX has been used for the display of Fv fragments (Gao et al., 1999). Furthermore, phage display has also been achieved on phage lambda. In that case, gene V protein (Maruyama et al., 1994), gene J protein, and gene D protein (Sternberg & Hoess, 1995; Mikawa et al., 1996) have been used.

[0003] Besides using genetic fusions, foreign peptides or proteins have been attached to phage surfaces via association domains. In WO 91/17271, it was suggested to use a tag displayed on phage and a tag binding ligand fused to the peptide/protein to be displayed to achieve a non-covalent display. A similar concept was pursued for the display of cDNA libraries (Crameri & Suter, 1993). There the jun/fos interaction was used to mediate the display of cDNA fragments. In their construct, additional cysteine residues flanking both ends of jun as well as fos further stabilised the interaction by forming two disulfide bonds

[0004] When screening phage display libraries in biopanning the problem remains how best to recover phage which have bound to the desired target. Normally, this is achieved by elution with appropriate buffers, either by using a pH-or salt gradient, or by specific elution using soluble target. However, the most interesting binders which bind with high affinity to the target might be lost by that approach. Several alternative methods have been devised which try to overcome that problem, either by providing a cleavage signal between the (poly)peptide/protein being displayed and its fusion partner, or between the target of interest and its carrier which anchors the target to a solid surface.

[0005] Furthermore, all the approaches referred to hereinabove require to use fision proteins comprising at least part of a phage coat protein and a foreign (poly)peptide/protein. Especially in the case of using gene III as partner for peptides/proteins to be displayed, this leads to several problems. First, the expression product of gene III is toxic to the host cell, which requires tight regulation of gene III fusion proteins. Second, expression of gene III products can make host cells resistant to infection with helper phage required for the production of progeny phage particles. And finally, recombination events between gene III fusion constructs and wild type copies of gene III lead to undesired artefacts. Furthermore, since at least the C-terminal domain of the gene III protein comprising about 190 amino acids has to be used in order to achieve incorporation of the fusion protein into the phage coat, the size of the vectors comprising the nucleic acid sequences is rather larger, leading to a decrease in transformation efficiency. Transformation efficiency, however, is a crucial factor for the production of very large libraries. Additionally, for the characterisation of (poly)peptide/proteins obtained after selection from a phage display library, the (poly)peptide/protein are usually recloned into expression vectors in order to remove the phage coat protein fusion partner, or in order to create new fusion proteins such as by fusion to enzymes for detection or to multimerisation domains. It would be advantageously to have a system which would allow direct expression without recloning, and direct coupling of the (poly)peptide/protein to other moieties.

[0006] Furthermore, most of these approaches (except for the work of Jespers et al. (1995), WO 91/17271, and Crameri & Suter (1993) mentioned hereinabove) are limited to the presentation of (poly)peptides/proteins having a free N-terminus, since the (poly)peptides/proteins have to be fused at the C-terminus with a phage coat protein. Especially in the case of cDNA libraries, or in the case of proteins requiring a free C-terminus to be functional, it would be highly desirable to have a simple method which doesn't require the generation of C-terminal fusions.

[0007] Thus, the technical problem underlying the present invention is to develop a simple, reliable system which enables the presentation of (poly)peptides/proteins on phage particles without the need to use fusion proteins with phage coat proteins. Additionally, there is a need for a method which allows to recover tightly binding (poly)peptides/proteins in a more reliable way. The solution to this technical problem is achieved by providing the embodiments characterised in the claims. Accordingly, the present invention allows to easily create and screen large libraries of (poly)peptides/proteins displayed on the surface of bacteriophage particles. The technical approach of the present invention, i.e. linking (poly)peptides/proteins by disulfide bonds to the surface of phage particles, is neither provided nor suggested by the prior art.

[0008] Thus, the present invention relates to a method for displaying a (poly)peptide/protein on the surface of a bacteriophage particle comprising:

[0009] causing or allowing the attachment of said (poly)peptide/protein after expression to a member of the protein coat of said bacteriophage particle, wherein said attachment is caused by the formation of a disulfide bond between a first cysteine residue comprised in said (poly)peptide/protein and a second cysteine residue comprised in said member of the protein coat.

[0010] In the context of the present invention, the term “bacteriophage” relates to bacterial viruses forming packages consisting of a protein coat containing nucleic acid required for the replication of the phages. The nucleic acid may be DNA or RNA, either double or single stranded, linear or circular. Bacteriophage such as phage lambda or filamentous phage (such as M13, fd, or f1) are well known to the artisan of ordinary skill in the art. In the context of the present invention, the term “bacteriophage particles” refers to the particles according to the present invention, i.e. to particles displaying a (poly)peptide/protein via a disulfide bonds. During the assembly of bacteriophages, the coat proteins may package different nucleic acid sequences, provided that they comprise a packaging signal. In the context of the present invention, the term “nucleic acid sequences” contained in bacteriophages or bacteriophage particles relates to nucleic acid sequences or vectors having the ability to be packaged by bacteriophage coat proteins during assembly of bacteriophages or bacteriophage particles. Preferably said nucleic acid sequences or vectors are derived from naturally occurring genomes of bacteriophage, and comprise for example, in the case of filamentous phage, phage and phagemid vectors. The latter are plasmids containing a packaging signal and a phage origin of replication in addition to plasmid features. The term “(poly)peptide” relates to molecules consisting of one or more chains of multiple, i. e. two or more, amino acids linked via peptide bonds. The term “protein” refers to (poly)peptides where at least part of the (poly)peptide has or is able to acquire a defined three-dimensional arrangement by forming secondary, tertiary, or quaternary structures within and/or between its (poly)peptide chain(s). This definition comprises proteins such as naturally occurring or at least partially artificial proteins, as well as fragments or domains of whole proteins, as long as these fragments or domains are able to acquire a defined three-dimensional arrangement as described above. Examples of (poly)peptides/proteins consisting of one chain are single-chain Fv antibody fragments, and examples for (poly)peptides/proteins consisting of more chains are Fab antibody fragments. When the first cysteine residue is located at the C-terminus of the (poly)peptide/protein, the display format corresponds to the conventional display set-up with the C-terminus being genetically fused to the member of the phage coat protein. However, by using the N-terminus of the (poly)peptide/protein, the display format can be reverted as in the pJuFO system of Crameri & Suter referred to above. The term “surface of a bacteriophage particle” refers to the part of a bacteriophage particle which is in contact with the medium the particle is contained in and which is accessible. The surface is determined by the proteins being part of the phage coat (the members of the protein coat of the particle) which is assembled during phage production in appropriate host cells. The term “after expression” refers to the situation that nucleic acid encoding said (poly)peptide/protein is expressed in a host cell prior to attachment of the (poly)peptide/protein to said coat, in contrast to approaches where nucleic acid encoding fusion proteins with bacteriophage coat proteins are being expressed. The expression of nucleic acid encoding said (poly)peptide/protein and the step of causing or allowing the attachment may be performed in separated steps and/or environments. Preferably, however, expression and the step of causing or allowing the attachment are being performed sequentially in an appropriate host cell. The term “wherein said attachment is caused by the formation of a disulfide bond” refers to a situation, wherein the disulfide bond is responsible for the attachment, and wherein no interaction domain for interaction with a second domain present in the (poly)peptide/protein has been recombinantly fused to said member of the protein coat, as for example in the case of the pJuFo system (Crameri & Suter, 1993).

[0011] In a preferred embodiment, the bacteriophage particle displaying the (poly)peptide/protein contains a nucleic acid sequence encoding the (poly)peptide/protein.

[0012] Methods for construction of nucleic acid molecules encoding a (poly)peptide/protein according to the present invention, for construction of vectors comprising said nucleic acid molecules, for introduction of said vectors into appropriately chosen host cells, for causing or allowing the expression of said (poly)peptides/proteins are well-known in the art (see, e.g., Sambrook et al., 1989; Ausubel et al., 1999; Ge et al, 1995). Further well-known are methods for the introduction of genetic material required for the generation of progeny bacteriophages or bacteriophage particles in appropriate host cells, and for causing or allowing the generation of said progeny bacteriophages or bacteriophage particles (see, e.g., Kay et al., 1996).

[0013] In a further preferred embodiment, the present invention relates to a method, wherein said second cysteine residue is present at a corresponding amino acid position in a wild type coat protein of a bacteriophage.

[0014] In a yet further preferred embodiment, the present invention relates to a method, wherein said member of the protein coat is a wild type coat protein of a bacteriophage.

[0015] The term “wild type coat protein” refers to those proteins forming the phage coat of naturally occurring bacteriophages. In the case of filamentous bacteriophage, said wild type proteins are gene III protein (pIII), gene VI protein (pVI), gene VII protein (pVII), gene VIII protein (pVIII), and gene IX protein (pIX). The sequences, including the differences between the closely related members of the filamentous bacteriophages such as f1, fd, and M13, are well known to one of ordinary skill in the art (see, e.g., Kay et al., 1996).

[0016] In a further preferred embodiment, said member of the protein coat is a truncated variant of a wild type coat protein of a bacteriophage, wherein said truncated variant comprises at least that part of said wild type coat protein causing the incorporation of said coat protein into the protein coat of the bacteriophage particle.

[0017] The term “truncated variant” refers to proteins derived from the wild type proteins referred to above which are modified by deletion of at least part of the wild type sequences. This comprises variants such as truncated gene III protein variants which have been found in bacteriophage mutants (Crissman & Smith, 1984) or which have been generated in the course of standard phage display methods (e.g. Bass et al., 1990; Krebber, 1996). For example, said truncated variant may consist, or include, the C-terminal domain of the gene III protein. To identify truncated variants according to the present invention, a detection tag may be fused to the variant, and an assay may be set up to determine whether the variant is incorporated into the phage coat of bacteriophage particles formed in the presence of the variant. By way of truncating a wild type protein by deleting a part of the wild type protein, a cysteine residue may become available which in the wild type protein was forming a disulfide bond with a second cysteine comprised in the deleted part.

[0018] In a yet further preferred embodiment, said member of the protein coat is a modified variant of a wild type coat protein of a bacteriophage, wherein said modified variant is capable of being incorporated into the protein coat of the bacteriophage particle.

[0019] Methods for achieving modification of a wild type protein according to the present invention are well-known to one of ordinary skill in the art, and involve standard cloning and/or mutagenesis techniques. Methods for the construction of nucleic acid molecules encoding a modified variant of a wild type protein used in a method according to the present invention, for construction of vectors comprising said nucleic acid molecules, including the construction of phage and/or phagemid vectors, for introduction of said vectors into appropriately chosen host cells, for causing or allowing the expression of said modified protein are well-known in the art (see, e.g., Sambrook et al., 1989; Ausubel et al., 1999; Kay et al., 1996). To identify modified variants according to the present invention, a detection tag may be fused to the variant, and an assay may be set up to determine whether the variant is capable or being incorporated into the phage coat of bacteriophage particles formed in the presence of the variant.

[0020] In a most preferred embodiment, said second cysteine residue is not present at a corresponding amino acid position in a wild type coat protein of a bacteriophage.

[0021] In a preferred embodiment, said second cysteine has been artificially introduced into a wild type coat protein of a bacteriophage.

[0022] In the context of the present invention, the term “artificially introduced” refers to a situation where a wild type coat protein has been modified by e.g. recombinant means. For example, nucleic acid encoding a wild type coat protein may be manipulated by standard procedures to introduce a cysteine codon creating a nucleic acid sequence encoding a modified coat protein, wherein a cysteine residue is artificially introduced by insertion into, or addition of said cysteine residue to, said at least part of a wild type or modified coat protein, or by substitution of an amino acid residue comprised in said at least part of a wild type or modified protein by said cysteine residue, or by fusion of said at least part of a wild type or modified coat protein with a (poly)peptide/protein comprising said second cysteine residue, or by any combination of said insertions, additions, substitutions or fusions. Upon expression of the nucleic acid comprising such recombinantly introduced cysteine codon, a variant of the wild type protein is formed comprising a cysteine residue.

[0023] In a further most preferred embodiment, said second cysteine has been artificially introduced into a truncated variant of a wild type coat protein of a bacteriophage.

[0024] In a yet further preferred embodiment, said second cysteine has been artificially introduced into a modified variant of a wild type coat protein of a bacteriophage.

[0025] Methods for achieving the artificial introduction according to the present invention are well-known to one of ordinary skill in the art, and involve standard cloning and/or mutagenesis techniques. Methods for the construction of nucleic acid molecules encoding a modified variant of a wild type protein used in a method according to the present invention, for construction of vectors comprising said nucleic acid molecules, for introduction of said vectors into appropriately chosen host cells, for causing or achieving the expression of said fusion proteins are well-known in the art (see, e.g., Sambrook et al., 1989; Ausubel et al., 1999).

[0026] In another embodiment, the present invention relates to a method, wherein said second cysteine is present at, or in the vicinity of, the C-or the N-terminus of said member of the phage coat of said bacteriophage particle.

[0027] The term “in the vicinity of” refers to a stretch of up to 15, or more preferably, up to 10 amino acids, counted in both cases from either N-or C-terminus of said (poly)peptide/protein, provided that the N-or C-terminus is located at the outside of the bacteriophage.

[0028] Yet further preferred is a method, wherein said bacteriophage is a filamentous bacteriophage. Filamentous bacteriophage such as M13, fd, or f1 are well known to the artisan of ordinary skill in the art.

[0029] In the case of filamentous bacteriophage, a method is particularly preferred, wherein said member of the protein coat of the bacteriophage particle is or is derived from the wild type coat protein pIII. Further preferred is a method, wherein said member of the protein coat of the bacteriophage particle is or is derived from the wild type coat protein pIX. In the context of the present invention, the term “is derived” refers to a modification, wherein the modified protein is capable of being incorporated into the protein coat of the bacteriophage particle. Preferably, those parts of the modified protein corresponding to the wild type protein exhibit an amino acid identity exceeding about 70%, preferably about 80%, most preferably about 90% compared to the corresponding wild type sequence.

[0030] In a yet further preferred embodiment of the present invention, the method comprises:

[0031] (a) providing a host cell harbouring a nucleic acid sequence comprising a nucleic acid sequence encoding said (poly)peptide/protein;

[0032] (b) causing or allowing the expression of said nucleic acid sequence; and

[0033] (c) causing or allowing the production of bacteriophage particles in said host cell.

[0034] In the context of the present invention, the term “causing or allowing the expression” describes cultivating host cells under conditions such that nucleic acid sequence is expressed. Methods for construction of nucleic acid molecules encoding a (poly)peptide/protein according to to the present invention, for construction of vectors comprising said nucleic acid molecules, for introduction of said vectors into appropriately chosen host cells, for causing or allowing the expression of (poly)peptides/proteins are well-known in the art (see, e.g., Sambrook et al., 1989; Ausubel et al., 1999). Further well-known are methods for the introduction of genetic material required for the generation of progeny bacteriophages or bacteriophage particles in appropriate host cells, and for causing or allowing the generation of said progeny bacteriophages or bacteriophage particles (see, e.g., Kay et al., 1996). The step of causing or allowing the production of bacteriophage particles may require the use of appropriate helper phages, e.g. in the case of working with phagemids.

[0035] The steps (b) and (c) may be performed sequentially, in either order, or simultaneously.

[0036] In a still further embodiment, said (poly)peptide/protein comprises an immunoglobulin or a functional fragment thereof.

[0037] In this context, “immunoglobulin” is used as a synonym for “antibody”. The term “functional fragment” refers to a fragment of an immunoglobulin which retains the antigen-binding moiety of an immunoglobulin. Functional immunoglobulin fragments according to the present invention may be Fv (Skerra & Plückthun, 1988), scFv (Bird et al., 1988; Huston et al., 1988), disulfide-linked Fv (Glockshuber et al., 1992; Brinkmann et al., 1993), Fab, F(ab′)₂ fragments or other fragments well-known to the practitioner skilled in the art, which comprise the variable domain of an immunoglobulin or immunoglobulin fragment. Particularly preferred is an scFv or Fab fragment.

[0038] In a preferred embodiment, the present invention relates to a nucleic acid sequence encoding a modified variant of a wild type coat protein of a bacteriophage, wherein said modified variant consists of:

[0039] (a) one or more parts of said wild type coat protein of a bacteriophage, wherein one of said parts comprises at least that part which causes or allows the incorporation of said coat protein into the phage coat; and

[0040] (b) between one and six additional amino acid residues not present at the corresponding amino acid positions in a wild type coat protein of a bacteriophage, wherein one of said additional amino acid residues is a cysteine residue.

[0041] In the context of the present invention, a modified variant obtained by substitution of an amino acid residue in a wild type coat protein sequence by a cysteine residue may be regarded as a variant composed of two parts of said wild type protein linked by an additional cysteine residue. Correspondingly, variants of a wild type coat protein comprising several mutations compared to the wild type sequence may be regarded as being composed of several wild type parts, wherein the individual parts are linked by the mutated residues. However, said variant may also result from the addition of up to six residues, including a cysteine residue, to either C-and or N-terminus of the wild type coat protein.

[0042] Further preferred is a nucleic acid sequence encoding a modified variant of a wild type coat protein of a bacteriophage, wherein said modified variant consists of:

[0043] (a) one or more parts of said wild type coat protein of a bacteriophage, wherein one of said parts comprises at least that part which causes or allows the incorporation of said coat protein into the phage coat;

[0044] (b) between one and six additional amino acid residues not present at the corresponding amino acid positions in a wild type coat protein of a bacteriophage, wherein one of said additional amino acid residues is a cysteine residue; and

[0045] (c) one or more peptide sequences for purification and/or detection purposes.

[0046] Particularly preferred are peptides comprising at least five histidine residues (Hochuli et at., 1988), which are able to bind to metal ions, and can therefore be used for the purification of the protein to which they are fused (Lindner et al., 1992). Also provided for by the invention are additional moieties such as the commonly used c-myc and FLAG tags (Hopp et al., 1988; Knappik & Plückthun, 1994), or the Strep-tag (Schmidt & Skerra, 1994; Schmidt et al., 1996).

[0047] The modified variant may further comprise amino acid residues required for cloning, for expression, or protein transport. Amino acid residues required for cloning may include residues encoded by nucleic acid sequences comprising recognition sequences for restriction endonucleases which are incorporated in order to enable the cloning of the nucleic acid sequences into appropriate vectors. Amino acid residues required for expression may include residues leading to increased solubility or stability of the (poly)peptide/protein. Amino acid residues required for protein transport may include signalling sequences responsible for the transport of the modified variant to the periplasm of E.coli, and/or amino acid residues facilitating the efficient cleavage of said signalling sequences. Further amino acid residues required for cloning, expression, protein transport, purification and/or detection purposes referred to above are numerous moieties well known to the practitioner skilled in the art.

[0048] In another embodiment, the present invention relates to a vector comprising a nucleic acid sequence according to the present invention.

[0049] In a preferred embodiment, the vector further comprises one or more nucleic acid sequences encoding a (poly)peptide/protein comprising a second cysteine residue.

[0050] In a most preferred embodiment, said (poly)peptide/protein comprises an immunoglobulin or a functional fragment thereof In the case of single-chain Fv antibody fragments referred to hereinabove, the vector comprises one nucleic acid sequence encoding the VH and VL domains linked by a (poly)peptide linker, and in the case of Fab antibody fragments, the vector comprises two nucleic acid sequences encoding the VH-CH and the VL-CL chains.

[0051] In a further embodiment, the present invention relates to a host cell containing a nucleic acid sequence according to the present invention or a vector according to the present invention.

[0052] In the context of the present invention the term “host cell” may be any of a number commonly used in the production of heterologous proteins, including but not limited to bacteria, such as Escherichia coli (Ge et al., 1995), or Bacillus subtilis (Wu et al., 1993), fungi, such as yeasts (Horwitz et al., 1988; Ridder et al., 1995) or filamentous fungus (Nyyssönen et al., 1993), plant cells (Hiatt & Ma, 1993; Whitelam et al., 1994), insect cells (Potter et al., 1993; Ward et al., 1995), or mammalian cells (Trill et al., 1995).

[0053] In a yet further preferred embodiment, the present invention relates to a modified variant of a wild type bacteriophage coat protein encoded by a nucleic acid sequence according to the present invention, a vector according to the present invention or produced by a host cell according to the present invention.

[0054] In another embodiment, the present invention relates to a bacteriophage particle displaying a (poly)peptide/protein on its surface obtainable by a method comprising: causing or allowing the attachment of said (poly)peptide/protein after expression to a member of the protein coat of said bacteriophage particle, wherein said attachment is caused by the formation of a disulfide bond between a first cysteine residue comprised in said (poly)peptide/protein and a second cysteine residue comprised in said member of the protein coat.

[0055] In another embodiment, the present invention relates to a bacteriophage particle displaying a (poly)peptide/protein attached to its surface, wherein said attachment is caused by the formation of a disulfide bond between a first cysteine residue comprised in said (poly)peptide/protein and a second cysteine residue comprised in a member of the protein coat of said bacteriophage particle.

[0056] In a preferred embodiment, the bacteriophage particle further contains a vector comprising one or more nucleic acid sequences encoding said (poly)peptide/protein.

[0057] In a most preferred embodiment of the present invention, the bacteriophage particle contains a vector according to the present invention, wherein said vector comprises a nucleic acid sequence encoding a modified wild type bacteriophage coat protein and furthermore one or more nucleic acid sequences encoding a (poly)peptide/protein and most preferably comprising at least a functional domain of an immunoglobulin.

[0058] The preferred embodiments of the method of the present invention referred to hereinabove mutatis mutandis apply to the bacteriophages of the present invention.

[0059] In a further embodiment, the present invention relates to a diverse collection of bacteriophage particles according to the present invention, wherein each of said bacteriophage particles displays a (poly)peptide/protein out of a diverse collection of (poly)peptides/proteins. A “diverse collection of bacteriophage particles” may as well be referred to as a “library” or a “plurality of bacteriophage particles”. Each member of such a library displays a distinct member of the library.

[0060] In the context of the present invention the term “diverse collection” refers to a collection of at least two particles or molecules which differ in at least part of their compositions, properties, and/or sequences. For example, a diverse collection of (poly)peptides/proteins is a set of (poly)peptides/proteins which differ in at least one amino acid position of their sequence. Such a diverse collection of (poly)peptides/proteins can be obtained in a variety of ways, for example by random mutagenesis of at least one codon of a nucleic acid sequence encoding a starting (poly)peptide/protein, by using error-prone PCR to amplify a nucleic acid sequence encoding a starting (poly)peptide/protein, or by using mutator strains as host cells in a method according to the present invention. These and additional or alternative methods for the generation of diverse collections of (poly)peptides/proteins are well-known to one of ordinary skill in the art. A “diverse collection of bacteriophage particles” may be referred to as a library or a plurality of bacteriophage particles. Each member of such a library displays a distinct member of the library.

[0061] In another embodiment, the invention relates to a method for obtaining a (poly)peptide/protein having a desired property comprising:

[0062] (a) providing the diverse collection of bacteriophage particles according to the present invention; and

[0063] (b) screening said diverse collection and/or selecting from said diverse collection to obtain at least one bacteriophage particle displaying a (poly)peptide/protein having said desired property.

[0064] In the context of the present invention the term “desired property” refers to a predetermined property which one of the (poly)peptides/proteins out of the diverse collection of (poly)peptides/proteins should have and which forms the basis for screening and/or selecting the diverse collection. Such properties comprise properties such as binding to a target, blocking of a target, activation of a target-mediated reaction, enzymatic activity, and further properties which are known to one of ordinary skill. Depending on the type of desired property, one of ordinary skill will be able to identify format and necessary steps for performing screening and/or selection.

[0065] Most preferred is a method, wherein said desired property is binding to a target of interest.

[0066] Said target of interest can be presented to said diverse collection of bacteriophage particles in a variety of ways well known to one of ordinary skill, such as coated on surfaces for solid phase biopanning, linked to particles such as magnetic beads for biopanning in solution, or displayed on the surface of cells for whole cell biopanning or biopanning on tissue sections. Bacteriophage particles having bound to said target can be recovered by a variety of methods well known to one of ordinary skill, such as by elution with appropriate buffers, either by using a pH-or salt gradient, or by specific elution using soluble target.

[0067] In a preferred embodiment, the method for obtaining a (poly)peptide/protein further comprises:

[0068] (ba) contacting said diverse collection of bacteriophage particles with the target of interest;

[0069] (bb) eluting bacteriophage particles not binding to the target of interest;

[0070] (bc) eluting bacteriophage particles binding to the target of interest by treating the complexes of target of interest and bacteriophages binding to said target of interest formed in step (ba) under reducing conditions. Under reducing conditions, such as by incubation with DTT, the disulfide bonds are cleaved, thus allowing to recover the specific bacteriophage particles for further rounds of biopanning and/or for identification of the (poly)peptide/proteins specifically binding to said target.

FIGURE LEGENDS

[0071]FIG. 1a: Vector Map of Construct pMorphX7-hag2-LH

[0072]FIG. 1b: Vector Sequence of pMorphX7-hag2-LH

[0073]FIG. 2: Vector Sequence of pTFT74-N1-hag-HIPM

[0074]FIG. 3: Vector Sequence of pQE60-MacI

[0075]FIG. 4: Specific Binding of scFv Displayed on Non-Engineered Phages.

[0076] Phages derived from constructs pMorphX7-MacI5-LCH, pMorphX7-MacI5-LHC and pMorphX7-MacI5-LH were produced by standard procedures and pre-incubated in PBSTM either with 5 mM DTT (+DTT) or without DTT. 5 μg/well of specific antigen (MacI, dark columns) as well as unspecific control antigen (BSA, light columns) were coated onto Maxisorp Nunc-Immuno microtiter plates and incubated with 1×10¹⁰ phages/well, respectively. Bound phages were detected via anti-M13-HRP conjugate and BM blue soluble substrate. Phages derived from conventional phage display vector pMorph13-MacI5 were used as control (3×10⁷ phages/well). Experimental details are given in Example 1.

[0077]FIG. 5: Specific Binding of scFv Displayed on Non-Engineered Phages

[0078] Phages derived from constructs pMorphX7-hag2-LCH, pMorphX7-hag2-LHC and pMorphX7-hag2-LH were produced by standard procedures and pre-incubated in PBSTM either with 5 mM DTT (+DTT) or without DTT. 5 μg/well of specific antigen (N1-hag, dark columns) as well as unspecific control antigen (BSA, light columns) were coated onto Maxisorp Nunc-Immuno microtiter plates and incubated with 1×10¹⁰ phages/well, respectively. Bound phages were detected via anti-M13-HRP conjugate and BM blue soluble substrate. Phages derived from conventional phage display vector pMorph13-hag2 were used as control (3×10⁷ phages/well). Experimental details are given in Example 1.

[0079]FIG. 6a: Vector Map of Construct pBR-C-gIII

[0080]FIG. 6b: Sequence of Expression Cassette for Full Length pIII With an N-Terminal Cysteine Residue (C-gIII)

[0081]FIG. 6c: Sequence of Expression Cassette for Truncated pIII With an N-Terminal Cysteine Residue (C-gIIICT)

[0082]FIG. 7a: Vector Map of Construct pMorph18-C-gIII- hag2-LHC

[0083]FIG. 7b: Vector Sequence of pMorph18-C-gIII-hag2-LHC

[0084]FIG. 8: Detection of scFv MacI-5 Displayed on Engineered hages-Two-Vector System

[0085] Phages derived from constructs pMorphX7-MacI-5-LH/pBR-C-gIII (lanes 1 & 5), pMorphX7-MacI-5-LHC/pBR-C-gIII (lanes 2 & 6), pMorphX7-MacI-5-LHC (lanes 3 & 7) and pMorphX7-MacI-5-LH (lanes 4 & 8) were produced by standard procedures. 1-5×10¹⁰ phages were pre-incubated in PBS with DTT (lanes 1-4) or without DTT (lanes 5-8). SDS loading buffer lacking reducing agents was added, phages were applied to an 4-15% SDS PAA Ready gel and analysed in immunoblots. Detection of scFvs associated with phages was done via anti-FLAG M1 antibody, anti-mouse-IgG-AP conjugate and Fast BCIP/NPT substrate (6A) and via anti-pIII antibody, anti-mouse-IgG-AP conjugate and Fast BCIPINPT substrate (6B). Low range marker (Amersham #RPN756) is marked as M. Experimental details are given in Example 2. 1.

[0086]FIG. 9: Detection of scFvs Displayed on Engineered Phages-One-Vector System

[0087] Phages derived from constructs pMorph18-C-gIII-hag2-LHC (lanes 1-8; 7A), pMorph18-C-gIII-AB1. 1-LHC (lanes 1, 2, 5 and 6; 7B) and pMorph18-C-gIII-MacI-5-LHC (lanes 3, 4, 7 and 8; 7B) were produced by standard procedures. 1-5×10¹⁰ phages were pre-incubated in PBS with DTT (lanes 1, 2, 5 and 6; 7A and lanes 1- 4; 7B) or without DTT (lanes 3, 4, 7 and 8; 7A and lanes 5-8; 7B). SDS loading buffer lacking reducing agents was added, phages were applied to an 4-15% SDS PAA Ready gel and analysed in immunoblots. Detection of scFvs associated with phages was done via anti-FLAG M1 antibody, anti-mouse-IgG-AP conjugate and Fast BCIP/NPT substrate (lanes 1-4; 7A) and via anti-pIII antibody, anti-mouse-IgG-AP conjugate and Fast BCIP/INPT substrate (lanes 5-8; 7A and lanes 1-8; 7B). Low range marker (Amersham #RPN756) is marked as M. Experimental details are given in Example 2.1.

[0088]FIG. 10: Specific Binding of scFv Displayed on Engineered Phages-Comparison of the Different Two-Vector Systems

[0089] Phages derived from constructs pMorphX7-MacI-5-LHC/pBR-C-gIII (1), pMorphX7-MacI-5-LHC/pBR-C-gIIICT (2), pMorphX7-MacI-5-LHC/pUC-C-gIII (3), pMorphX7-MacI-5-LHC/pUC-C-gIIICT (4), pMorphX7-MacI-5-LHC (5), pMorphX7-MacI-5-LH (6) and the conventional phage display vector pMorph13-MacI-5 (7) were produced by standard procedures. 5 μg of specific antigen (MacI) as well as unspecific control antigen (BSA, data not shown) were coated onto Maxisorp Nunc-Immuno microtiter plates and incubated with a range of 6.4×10⁶ and 1×10¹¹ phages per well. Bound phages were detected via anti-M13-HRP conjugate and BM blue substrate. Experimental details are given in Example 2. 1

[0090]FIG. 11: Specific Binding of scFv Displayed on Engineered Phages-Comparison of the One-and Two-Vector System

[0091] Phages derived from constructs pMorphX7-MacI-5-LHC/pBR-C-gIII (1), pMorphX7-MacI-5-LHC/pBR-C-gIIICT (2), pMorph18-C-gIII-MacI-5-LHC (3), pMorph18-C-gIIICT-MacI-5-LHC (4) and pMorphX7-MacI-5-LHC (5) were produced by standard procedures. 5 μg of specific antigen (MacI, dark columns) as well as unspecific control antigen (BSA, light columns) were coated onto Maxisorp Nunc-Immuno microtiter plates and incubated with 1×10¹⁰ and 1×10⁹ phages, respectively. Bound phages were detected via anti-M13-HRP conjugate and BM blue substrate. Experimental details are given in Example 2.1.

[0092]FIG. 12: Specific Binding of scFv Displayed on Engineered Phages-Comparison of Engineered Gene III and Gene IX Proteins in the One-Vector System

[0093] Phages derived from constructs pMorph18-C-gIII-MacI-5-LHC (1), pMorph18-C-gIIICT-MacI-5-LHC (2), pMorph18-C-gIX-MacI-5-LHC (3), pMorphX7-MacI-5-LHC (4) and the conventional phage display vector pMorph13-MacI-5 (5) were produced by standard procedures. 5 μg of specific antigen (MacI, dark columns) as well as unspecific control antigen (BSA, light columns) were coated onto Maxisorp Nunc-Immuno microtiter plates and incubated with 1×10¹⁰, 1×10⁹ and 1×10⁸ phages, respectively. Bound phages were detected via anti-M13-HRP conjugate and BM blue substrate. Experimental details are given in Example 2.1.

[0094]FIG. 13: Specific Binding of scFv Displayed on Engineered Phages-Impact of DTT

[0095] Phages derived from constructs pMorph18-C-gIII-MacI-5-LHC (1), pMorph18-C-gIIICT-Mac1-5-LHC (2), pMorph18-C-gIX-MacI-5-LHC (3), pMorphX7-MacI-5-LHC (4) and the conventional phage display vector pMorph13-MacI-5 (5) were produced by standard procedures and pre-incubated in PBSTM either with 5 mM DTT (+) or without DTT (−). 5 μg of specific antigen (MacI, dark columns) as well as unspecific control antigen (BSA, light columns) were coated onto Maxisorp Nunc-Immuno microtiter plates and incubated with 1×10¹⁰ phages respectively. Bound phages were detected via anti-M13-IP conjugate and BM blue substrate. Experimental details are given in Example 2.1.

[0096]FIG. 14: Specificity of Selected scFvs-Panning of Pre-Selected Pools Against N1-MacI

[0097] scFvs selected after two rounds of cys-display panning against antigen N1-MacI from the κ-chain (1-5) and the λ-chain pool (6-8) were expressed according to standard procedures. 0.1 μg/well of milk powder (A), BSA (B), FITC-BSA (C, FITC coupled to BSA), N1-hag (D), N1-Np50 (E) and N1-MacI (N1-MacI) was coated onto 384 well plates (Maxisorp; Nunc) and incubated with 10 μl scFv solution, respectively. Bound scFvs were detected via a mixture of anti-Flag M1, anti-Flag M2 and anti-mouse IgG-AP conjugate as well as AttoPhos fluorescence substrate (Roche #1484281). Each scFv was tested in quadruplicates and mean values are presented.

[0098]FIG. 15: Specificity of Selected scFvs-Panning of Pre-Selected Pools Against N1-Np50

[0099] scFvs selected after two rounds of cys-display panning against antigen N1-Np50 (1-8) were expressed according to standard procedures. 0.1 μg/well of milk powder (A), BSA (B), FITC-BSA (C, FITC coupled to BSA), N1-hag (D), N1-MacI (E) and N1-Np50 (N1-Np50) was coated onto 384 well plates (Maxisorp; Nunc) and incubated with 10 μl scFv solution, respectively. Bound scFvs were detected via a mixture of anti-Flag M1, anti-Flag M2 and anti-mouse IgG-AP conjugate as well as AttoPhos fluorescence substrate (Roche #1484281). Each scFv was tested in quadruplicates and mean values are presented.

[0100]FIG. 16a: Vector Map of Construct pMorphX10-Fab-MacI5-VL-LHC-VH-FS

[0101]FIG. 16b: Complete Vector Sequence of pMorphX10-Fab-MacI5-VL-LHC-VH-FS

[0102]FIG. 17: Detection of Fab ICAM1-C8 Displayed on Engineered Phages

[0103] Phages derived from constructs pMorphX10-Fab-ICAM1C8-VL-LHC-VH-MS/pBAD-SS-C-gIII (lanes 5,6,11,12), pMorphX10-Fab-ICAM1C8-VL-LHC-VH-MS (lanes 3,4,9,10) and pMorph18-Fab-ICAM1C8 (lanes 1,2,7,8) were produced by standard procedures. 1×10¹⁰ phages were pre-incubated in PBS with DTT (lanes 1-6) or without DTT (lanes 7-12). SDS loading buffer lacking reducing agents was added, phages were applied to an 12 % SDS PAA Ready gel and analysed in immunoblots. Detection was done via anti-pIII antibody, anti-mouse-IgG-HRP conjugate and BM Blue POD precipitating substrate. Low range molecular weight marker (Amersham Life Science #RPN756) is marked as M. Experimental details are given in Example 2.2.

[0104]FIG. 18: Detection of Fab MacI-A8 Displayed on Engineered Phages

[0105] Phages derived from constructs pMorphX10-Fab-MacIA8-VL-LHC-VH-FS/pBAD-SS-C-gIII (lanes 5,6,11,12), pMorphX10-Fab-MacIA8-VL-LHC-VH-FS (lanes 3,4,9,10) and pMorph18-Fab-MacIA8 (lanes 1,2,7,8) were produced by standard procedures. 1×10¹⁰ phages were pre-incubated in PBS with DTT (lanes 1-6) or without DTT (lanes 7-12). SDS loading buffer lacking reducing agents was added, phages were applied to an 12% SDS PAA Ready gel and analysed in immunoblots. Detection was done via anti-pIII antibody, anti-mouse-IgG-HRP conjugate and BM Blue precipitating substrate. Low range molecular weight marker (Amersham Life Science #RPN756) is marked as M. Experimental details are given in Example 2.2.

[0106]FIG. 19: Specific Binding of Fabs Displayed on Engineered Phages-Fab MacI-5

[0107] Phages derived from constructs pMorphX10-Fab-MacI5-VL-LHC-VH-FS/pBR-C-gIII (1), pMorphX10-Fab-MacI5-VL-C-VH-FS/pBR-C-gIII (2), pMorphX10-Fab-MacI5-VL-VH-CFS/pBR-C-gIII (3), pMorphX10-Fab-MacI5-VL-VH-LHC/pBR-C-gIII (4), pMorphX9-Fab-MacI5-FS (5), and the conventional phage display vector pMorph18-Fab-MacI5 (6) were produced by standard procedures. 5 μg/well of specific antigen (N1-MacI) were coated onto Maxisorp Nunc-Immuno microtiter plates and incubated with 1×10⁸ (light columns) and 1×10⁹ (dark columns) phages per well. Bound phages were detected via anti-M13-HRP conjugate and BM blue soluble substrate. Each column represents the mean value of three independent phage preparations tested in duplicates. Experimental details are given in Example 2.2.

[0108]FIG. 20: Specific Binding of Fabs Displayed on Engineered Phages-Fab MacI-A8

[0109] Phages derived from constructs pMorphX10-Fab-MacIA8-VL-LHC-VH-FS/pBR-C-gIII (1), pMorphX10-Fab-MacIA8-VL-C-VH-FS/pBR-C-gIII (2), pMorphX10-Fab-MacIA8-VL-VH-CFS/pBR-C-gIII (3), pMorphX10-Fab-MacIA8-VL-VH-LHC/pBR-C-gIII (4), pMorphX9-Fab-MacIA8-FS (5), and the conventional phage display vector pMorphl8-Fab-MacIA8 (6) were produced by standard procedures. 5 μg/well of specific antigen (N1-MacI) were coated onto Maxisorp Nunc-Immuno microtiter plates and incubated with 1×10⁹ (light columns) and 1×10¹⁰ (dark columns) phages per well. Bound phages were detected via anti-M13-HRP conjugate and BM blue soluble substrate. Each column represents the mean value of three independent phage preparations tested in duplicates. Experimental details are given in Example 2.2.

[0110]FIG. 21: Specific Binding of Fabs Displayed on Engineered Phages-Fab ICAM1-C8

[0111] Phages derived from constructs pMorphX10-Fab-ICAM1C8-VL-LHC-VH-MS/pBR-C-gIII (1), pMorphX10-Fab-ICAM1C8-VL-C-VH-MS/pBR-C-gIII (2), pMorphX10-Fab-ICAM1C8-VL-VH-CMS/pBR-C-gIII (3), pMorphX10-Fab-ICAM1C8-VL-VH-LHC/pBR-C-gIII (4), pMorphX9-Fab-ICAM1C8-MS (5), pMorphX9-Fab-ICAM1C8-MS/pBR-C-gIII (6) were produced by standard procedures. 5 μg/well of specific antigen (ICAM1, dark columns) or unspecific antigen (BSA, light columns) were coated onto Maxisorp Nunc-Immuno microtiter plates and incubated with 1×10⁹ phages per well. Bound phages were detected via anti-M13-HRP conjugate and BM blue soluble substrate. Each column represents the mean value of one phage preparation tested in duplicates. Experimental details are given in Example 2.2.

[0112]FIG. 22: Specific Binding of Fabs Displayed on Engineered Phages-Impact of DTT

[0113] Phages derived from constructs pMorphX10-Fab-MacI5-VL-LHC-VH-FS/pBR-C-gIII (1), pMorphX10-Fab-MacI5-VL-C-VH-FS/pBR-C-gIII (2), pMorphX10-Fab-MacI5-VL-VH-CFS/pBR-C-gIII (3), pMorphXio-Fab-MacI5-VL-VH-LHC/pBR-C-gIII (4), pMorphX9-Fab-MacI5-FS (5), and the conventional phage display vector pMorphl8-Fab-MacI5 (6) were produced by standard procedures and pre-incubated in PBSTM either with 10 MM DTT (+) or without DTT (−). 5 μg/well of specific antigen (N1-MacI, dark columns) as well as unspecific control antigen (BSA, light columns) were coated onto Maxisorp Nunc-Immuno microtiter plates and incubated with 1×10⁹ phages respectively. Bound phages were detected via anti-M13-HRP conjugate and BM blue substrate. Each column represents the mean value of one phage preparation tested in duplicates. Experimental details are given in Example 2.2.

[0114] The examples illustrate the invention.

EXAMPLE 1

[0115] Display of (Poly)Peptides/Proteins on the Surface of Non-Engineered Filamentous Bacteriophage Particles Via Formation of Disulfide Bonds

[0116] In the following example, all molecular biology experiments are performed according to standard protocols (Ausubel et al., 1999).

[0117] Construction of Vectors Expressing scFvs

[0118] All vectors used are derivatives of the high copy phagemid pMorphX7-LH (FIG. 1a+b), a derivative of the pCAL vector series (WO 97/08320; Knappik et al., 2000). The expression cassette comprises the phoA signal sequence, a minimal binding site for the monoclonal antibody (mab) anti-FLAG M1 (Sigma #F-3040) (Knappik and Plückthun, 1994), a single chain fragment (scFv), a short linker (PGGSG) and a 6× histidine tag (6His; Hochuli et al., 1988) (FIG. 1a). pMorphX7-LCH and pMorphX7-LHC have been generated by inserting oligonucleotide cassettes coding for Cys-6His and 6His-Cys, respectively, between the unique AscI and HindlII sites of pMorphX7-LH (FIG. 1a, Table 1). All vectors express soluble scFv not genetically fused to any phage coat protein. The conventional phage display vector pMorph13 which is based on the pCAL4 vector described in WO 97/08320 and expresses a fusion of an scFv to the C-terminal part of phage protein pIII was used as positive control. The scFvs have been exchanged between the respective vectors via the unique XbaI and EcoRI sites (c.f. FIG 1 a).

[0119] Description of the scfv-Antigen Interactions

[0120] All scFvs derive from a human combinatorial antibody library (HuCAL; WO 97/08320; Knappik et al., 2000). The HuCAL VH and VL consensus genes (described in WO 97/08320), and the CDR3 sequences of the scFvs are given in Table 2. Clone hag2 was selected against a peptide from influenza virus hemagglutinine (aa 99-110 from hemagglutinine plus additional flanking aa (shown in italics, CAGPYDVPDYASLRSHH (SEQ ID NO: 14)), and clone MacI-5 against a fragment (MacI) of human CR-3 alpha chain (SWISS-PROT entry P11215, aa 149-353 of human CR-3 alpha fused to a C-terminal sequence containing a 633 histidine tag). The corresponding antigens for ELISA and doped library experiments were obtained as follows. The hag2 specific antigen N1-hag was produced using expression vector pTFT74-N1-hag-HIPM, a derivative of vector pTFT74 (Freund et al., 1993) (FIG. 2). N1-hag comprises aa 1-82 of mature gene III protein of phage M13 containing an additional methionine residue at the N-terminus (N1) fused to the amino acid sequence PYDVPDYASLRSHHHHHH (hag) (SEQ ID NO:1) comprising aa 99-110 from influenza virus hemagglutinine and a 6× histidine tag (in italics). Expression, purification and refolding of Ni-hag was done as described (Krebber, 1996; Krebber et al., 1997). As antigen for MacI-5, a purified fragment (MacI) of human CR-3 alpha chain (SWISS-PROT entry P11215) fused to a C-terminal 6× histidine tag was used. In detail, the expression cassette encodes an N-terminal methionine, amino acids 149 - 353 of human CR-3 alpha and amino acids IEGRHHHHHH (SEQ ID NO:2). This cassette is flanked by unique restriction sites BspHI and HindIII and can e.g. be introduced into the unique NcoI and HindIII sites of pQE-60 (QIAGEN GmbH, Hilden, Germany), yielding expression vector pQE60-MacI (FIG. 3). Expression and purification was performed using standard methods (The QIAexpressionist™ 3rd edition: A handbook for high-level expression and purification of 6×His-tagged proteins (July 1998). QIAGEN GmbH, Hilden, Germany). Bovine serum albumin (BSA, Sigma #A7906) was used as negative control antigen.

[0121] Functionality of scFvs Displayed on Non-Engineered Phages

[0122] To demonstrate that the displayed scFvs are functional with respect to recognition of their specific antigens phage ELISAs were performed. The analysis was done for the two HuCAL scFvs hag2 and MacI-5. Three expression systems differing in the modules fused to the C-terminus of the scFv were analysed, namely pMorphX7-LH, pMorphX7-LHC and pMorphX7-LCH.

[0123] Phages were produced according to standard procedures using helper phage VCSM13 (Kay et al., 1996). Specific antigen or control antigen (BSA, Sigma #A7906) was coated for 12 h at 4° C. at a concentration of 5 μg/well in PBS to Nunc Maxisorp microtiter plates (# 442404).

[0124] Phages were pre-incubated in PBSTM (PBS containing 5% skimmed milk powder and 0.1% Tween 20), either with or without 5 mM DTT, for 2 h at room temperature before they were applied to the ELISA well coated with antigen at a concentration of 1×10¹⁰ phages per well except for pMorph13 which was used at a concentration of 3×10⁷ phages per well. After binding for 1 h at RT, non-specifically bound phages were washed away with PBS containing 0.05% Tween 20 and bound phages were detected in ELISA using an anti-M13-HRP conjugate (Amersham Pharmacia Biotech #27-9421-01) and BM blue soluble (Boehringer Mannheim #1484281). Absorbance at 370 nm was measured. ELISA signals obtained with the specific antigen were compared to those with the control antigen. Specific binding of scFv displaying phages to antigen could be shown. As an example two of such ELISAs for scFvs hag2 and MacI-5 are presented in FIGS. 4 and 5, respectively. With phages derived from pMorphX7-LCH and pMorphX7-LHC signals between 1.9 and 5.8 times above background were achieved. When 5 mM DTT was added to the phages prior to antigen binding during the pre-incubation step, the ELISA signal was decreased to almost background levels while DTT had no major effect on the conventional display phages (pMorph13).

[0125] Enrichment of Non-Engineered Phages Displaying scFv

[0126] To prove that non-engineered phages displaying scFvs can be enriched on specific antigen a so called doped library experiment was performed. Specific phages were mixed with a high excess of unspecific phages and three rounds of panning on specific antigen were performed. The enrichment for specific phages was determined after each round. The analysis was done for the two HuCAL scFvs hag2 and MacI-5 in the pMorphX7-LHC vector.

[0127] pMorphX7-hag2-LHC and pMorphX7-MacI-5-LHC derived phages were mixed at ratios of 1:10⁵ (pMorphX7-hag2-LHC panning) as well as 10⁵:1 (pMorphX7-MacI-5-LHC panning). Three rounds of panning were performed on the hag2 and MacI-5 specific antigen, respectively. Phages were prepared by standard procedure and pre-blocked by mixing 1:1 with PBSTM (PBS, 5% skimmed milk powder, 0.1% Tween20) and incubation for 2 h at RT. Wells of a Nunc Maxisorp microtiter plate (#442404) were coated with specific antigen Ni -hag (as well as BSA) at a concentration of 5 μg/well in PBS overnight at 4° C., and subsequently blocked with 400 μl PBSM (PBS, 5% skimmed milk powder) for 2 h at RT. For the first round, 10¹¹ pre-blocked phages were applied per well and incubated for 1 h at RT on a microtiter plate shaker. Phage solution was removed and wells were washed 3 times with PBST (PBS, 0.05% Tween20) and 3 times with PBS. Bound phages were eluted with 100 mM triethylamine according to standard protocols and used for infection of TG1 cells. In addition, residual phages were eluted by direct infection of TG1 added to the wells. After each round of panning on specific antigen the ratio of specific to unspecific phages was determined by analysing at least 46 independent infected cells via PCR. The PCR was performed according to standard protocols using single colonies as source of template and oligonucleotides specific for VH CDR3 and VL CDR3 of each scFv as primers. After 3 rounds of panning, ˜4% positive clones (4 out of 93 clones analysed) were obtained for the pMorphX7-hag2-LHC panning and ˜90% positive clones (82 out of 91 clones analysed) were obtained for the pMorphX7-MacI-5-LHC panning.

EXAMPLE 2

[0128] Display of (Poly)Peptides/Proteins on the Surface of Engineered Filamentous Bacteriophage Particles Via Formation of Disulfide Bonds

EXAMPLE 2.1

[0129] Display of scFvs

[0130] Example 1 described above shows that functional scfvs can be displayed on non-engineered phages via disulfide bonds. This system can be further improved, e.g. via engineering an exposed cysteine on a phage coat protein. One candidate phage coat protein is protein III (pIII) which is composed of three domains N1, N2 and pIIICT. Possible sites for positioning an unpaired cysteine residue are the linker regions between the domains or the exposed N-terminus of the domain or the pIIICT in a truncated pIII version. A further example would be phage coat protein IX (pIX) where the cysteine could e.g. be linked to the N-terminus of the full length protein. In principle the cassettes for expression of such engineered proteins can be placed on the vector which is providing the scFv (one-vector system), or on a separate vector (two-vector system).

[0131] In the following we will describe experiments in which we engineered both a full length and a truncated pIII version as well as pIX) These proteins were co-expressed in the same bacterial cell together with the scfv, either from the same phagemid (pMorph18-C-gIII-scFv-LHC derivatives; one-vector system) or from a separate plasmid (pBR322-C-gIII or pUC19-C-gIII and derivatives; two-vector system).

[0132] Construction of Vectors Expressing scFvs and Engineered Phage Coat Proteins

[0133] Phage coat protein expression cassettes for the two-vector system were constructed as follows: Two different expression cassettes flanked by unique NheI and HindIII restriction sites at the ends were made positioning an unpaired cysteine residue at the exposed N-terminus of the N1-domain of full length mature pIII (C-gIII) or at the N-terminus of the pIIICT domain of the truncated protein (amino acids 216 to 406 of protein pIII; C-gIIICT) (FIG. 6b+c)). Both expression cassettes are under the control of the lac promotor/operator region and comprise the signal sequence ompA, amino acids DYCDIEF (SEQ ID NO:3) and the pIII or pIIICT ORF (complete amino acid sequences are given in Table 3). Plasmids expressing the modified pIII proteins were obtained by inserting these NheI-HindIII cassettes into plasmid pBR322 and pUC19 via the unique NheI and HindIII or XbaI and HindIII sites, respectively. As an example, the vector map of pBR-C-gIII is depicted in FIG. 6a. The resulting plasmids, pBR-C-gIII, pBR-C-gIIICT, pUC-C-gIII and pUC-C-gIIICT, were co-transformed with pMorphX7-LHC phagemids expressing the modified scFv (Example 1) into E.coli TG1 selecting for both antibiotic markers.

[0134] In the one-vector system both the modified phage coat proteins as well as the modified scFv were expressed from a dicistronic phagemid under control of the lac promotor/operator region. The first expression cassette comprises the signal sequence ompA, amino acids DYCDIEF (SEQ ID NO:3) and the ORF for the respective phage coat protein or part thereof. The unpaired cysteine residue was linked to the exposed N-terminus of the N1-domain of full length mature pIII (C-gIII), to the N-terminus of the truncated protein III (amino acids 216 to 406 of protein pIII; C-gIIICT) and to the N-terminus of protein IX (C-gIX), respectively (amino acid sequences are given in Table 4). The second expression cassette comprises the phoA signal sequence, the ORF of the respective scFv, a short linker (PGGSG), a 6× histidine tag (6His; Hochuli et al., 1988) and the single cysteine residue (see pMorphX7-LHC, Table 1). The complete vector sequence of pMorph18-C-gIII-hag2-LHC coding for modified full length pll as well as modified scFv hag2 and the respective vector map are given in FIG. 7a+b. The different phage coat proteins can be exchanged via EcoRI and StuI in a three fragment cloning procedure due to a second EcoRI site at the 3′ end of the scFvs. The different engineered scFvs can be cloned via the unique MfeI and HindIII sites. A derivative of this vector, pMorph20-C-gIII-hag2-LHC, contains a unique EcoRI site at the 3′ end of the scFv while the second site (between the ompA signal sequence and the gIII ORF) was deleted via silent PCR mutagenesis. This construct allows the cloning of scFvs or scFv pools via the unique SphI and EcoRI sites.

[0135] Attachment of scFvs to Phage Coat Proteins via Disulfide Bonds

[0136] Phage for biopanning applications can be produced using helper phage VCSM13 following standard protocols (Kay et al., 1996). In addition to helper phage proteins, engineered phage coat protein and soluble modified scFv were co-expressed from the one-or two-vector systems described above. To demonstrate that the scFvs attach to the engineered phage coat proteins via disulfide bridges and are incorporated into phage particles, scFv displaying phages were run on SDS PAGE under non-reducing and reducing conditions. Western blot analysis was performed with anti-pIII and anti-Flag M1 antisera.

[0137] Phages were produced according to standard procedures using helper phage VCSM13 (Kay et al., 1996). Phages were pre-incubated in PBS with 5 mM DTT or without DTT (reducing and non-reducing conditions, respectively) for 30 minutes at room temperature before adding SDS loading buffer lacking reducing agents such as DTT or β-mercaptoethanol. 1-5×10¹⁰ phages per lane were run on a 4-15% SDS PAGE (BioRad) and blotted onto PVDF membranes. For the anti-pIII Western blot, the membrane was blocked in MPBST (PBS buffer containing 5% milk powder and 0.05% Tween20) and developed with mouse anti-pIII (1:250 dilution; Mobitec) as primary antibody, anti-mouse-IgG-AP conjugate (1:10000 dilution; SIGMA) as secondary antibody and BCIP/NPT tablets (SIGMA) as substrate. For the anti-Flag M1 Western blot, the membrane was blocked in MTBST- CaCl₂ (TBS buffer containing 5% milk powder, 0.05% Tween20 and 1 mM CaCl₂) and developed with mouse anti-Flag M1 (1:5000 dilution; Sigma) as primary antibody, anti-mouse-IgG-AP conjugate (1:10000 dilution; SIGMA) as secondary antibody and BCIP/NPT tablets (SIGMA) as substrate.

[0138] Specific bands migrating at the height expected for the scFv linked to the full length pIII could be shown both for the one-and two-vector system. This signal can only be seen under non-reducing conditions and disappears under DTT indicating that pIII and scFv are linked via disulfide bonds (scFv-S-S-pIII). As an example for the two-vector system an anti-Flag M1 and anti-pIII Western blot for scFv MacI-5 is shown in FIG. 8. When the scFv without additional cysteines (pMorph7x-MacI-5-LH) is expressed, only free scFv sticking to phages can be detected in the anti-Flag M1 Western blot (lane 8, FIG. 8A). When an additional cysteine is added to the scFv (pMorphX7-MacI-5-LHC), those bands can hardly be seen and a band migrating at the height of scFv dimers (scFv-S-S-scFv and/or (scFv-SH)₂) (and an unknown additional band (scFv-S-SX)) appear (lane 7, FIG. 8A). When the engineered scFvs are co-expressed with an engineered pIII containing an additional cysteine at the N-terminus (pMorphX7-MacI-5-LHC and pBR-C-gIII) the signals shift to a molecular weight corresponding to scFv-pIII heterodimers (scFv-S-S-pIII) (lane 6, FIG. 8A). As expected, this scFv-S-S-pIII signal cannot be seen when non-engineered scFvs are co-expressed with the engineered pIII (pMorphX7-MacI-5-LH and pBR-C-gIII), although similar numbers of phage particles are loaded in each lane (lane 5, FIG. 8A). In the presence of reducing agents, the predominant signals are obtained from free scFvs for all expression systems (lanes 1 -4, FIG. 8A). In the anti-pIII Western blot, free protein III (pIII-SH and/or pIII) can be seen for all expression systems both under reducing and non-reducing conditions (lanes 1 -8, FIG. 8B). Specific bands migrating at the height expected for disulfide bonded protein III dimers (pIII-S-S-pIII) can only be detected under non-reducing conditions when engineered protein III is expressed (lanes 5 and 6 of FIG. 8B). Only when both engineered scFv and engineered protein III are co-expressed an additional band migrating at the height of a disulfide-linked scFv and protein III (scFv-S-S-pIII) appears in addition to the disulfide bonded protein III dimers (lane 6, FIG. 8B). This band corresponds in size to the scFv-S-S-pIII signal detected in the anti-Flag M1 Western (c.f. lane 6, FIG. 8A) and is DTT sensitive (c.f lane 2, FIG. 8A). DTT sensitive bands migrating at the height of disulfide-linked scFv and protein III and being detected both with anti-Flag M1 and anti-pIII antisera were also observed when engineered scFv and engineered pIII were co-expressed from the same phagemid (pMorphl8-C-pIII-scFv-LHC). As an example for this one-vector system an anti-Flag M1 and anti-pIII Western blot for scFv hag2 and anti-pIII Western blots for scFvs AB 1.1 and MacI-5 are shown in FIGS. 9A and 9B, respectively.

[0139] Functionality of scFvs Displayed on Engineered Phages

[0140] To show that the displayed scFvs are functional with respect to recognition of the specific antigen, phage ELISAs were performed. The analysis was done for the HuCAL scFvs MacI-5 and hag2. For the two-vector system, pMorphX7-LHC was co-transformed with pBR-C-gIII, pBR-C-gIIICT, pUC-C-gIII and pUC-C-gIIICT, respectively. Three different one-vector constructs were analysed, namely pMorph18-C-gIII-scFv-LHC, pMorph18-C-gIIICT-scFv-LRC and pMorph18-C-gIX-scFv-LHC. To demonstrate that the scFvs attach to the engineered phage coat proteins via disulfide bonds, phage ELISAs were performed both under non-reducing and reducing conditions.

[0141] Phages were produced according to standard procedures using helper phage VCSM13 and phage titers were determined (Kay et al., 1996). Specific antigen or control antigen (BSA, Sigma #A7906) was coated for 12 hours at 4° C. at an amount of 5 μg/well in PBS to Nunc Maxisorp microtiter plates (# 442404) and blocked with PBS containing 5% skimmed milk powder for 2 h. Phages were pre-incubated in PBS containing 2.5% skimmed milk powder, 0.05% Tween 20, as well as 5 mM DTT, where applicable, for 2 h at room temperature before they were applied to the ELISA well coated with antigen at a concentration range between 6.4 ×10⁶ and 1×10¹¹ phages per well. After binding for 1 h at RT, unspecifically bound phages were washed away with PBS containing 0.05% Tween 20 and bound phages were detected in ELISA using an anti-M13HRP conjugate (Amersham Pharmacia Biotech #27-9421-01) and BM blue soluble (Boehringer Mannheim #1484281). Absorbance at 370 nm was measured. ELISA signals obtained with the specific antigen were compared to those with the control antigen. Specific binding of scFv displaying phages to antigen could be shown for the C-gIII, C-gIIICT and C-gIX constructs in the one-vector format. C-gIII and C-gIIICT were also tested and shown to work in both two-vector systems. As an example four such ELISAs for scFv MacI-5 are presented in FIGS. 10-13. In all cases where phage coat proteins are engineered with an additional cysteine residue, ELISA signals are significantly increased compared to the pMorphX7-LHC signals where only the scFv carries an additional cysteine. When 5 mM DTT was added to the phages prior to antigen binding during the pre-incubation step, the ELISA signal was decreased to almost background levels for all three engineered phage coat constructs as well as the non-engineered pMorphX7-LHC phages while DTT had no major effect on the conventional display phages (pMorph13; FIG. 13). This shows that for both the non-engineered and engineered phages disulfide bonds are essential for the functional display of scFvs on phages and thus for the specific binding of scFv displaying phages to antigen.

[0142] Enrichment of Engineered Phages Displaying scfv in “Doped Library” Experiments

[0143] To prove that engineered phages displaying scFvs can be enriched on specific antigen, a “doped library” experiment was performed: specific phages were mixed with a high excess of unspecific phages and three rounds of panning on specific antigen were performed The enrichment for specific phages was determined after each round. The analysis was done for the two HuCAL scFvs hag2 and MacI-5 in the pMorph18-C-gIII-scFv-LHC one-vector system.

[0144] pMorph18-C-gIII-hag2-LHC and pMorph18-C-gIII-MacI-5-LHC derived phages were mixed at ratios of 1:10 (pMorph18-C-gIII-hag2-LHC panning) as well as 10⁵:1 (pMorph18-C-gIII-MacI-5-LHC panning). Three rounds of panning were performed on the hag2 and MacI-5 specific antigen, respectively. Phages were prepared by standard procedure and pre-blocked by mixing 1:1 with PBSTM (PBS, 5% skimmed milk powder, 0.1% Tween20) and incubation for 2 h at RT Wells of a Nunc Maxisorp plate (#442404) were coated with specific antigen (as well as BSA) at a concentration of 5 μg/well in PBS overnight at 4° C., and subsequently blocked with 400 μl PBSM (PBS, 5% skimmed milk powder) for 2 h at RT. For the first round, 10¹⁰ pre-blocked phages were applied per well and incubated for 1 h at RT on a microtiter plate shaker. Phage solution was removed and wells were washed 3 times with PBST (PBS, 0.05% Tween20) and 3 times with PBS. Bound phages were eluted with 100 mM triethylamine according to standard protocols and used for infection of TG1 cells. In addition, residual phages were eluted by direct infection of TG1 cells added to the wells. After each round of panning on specific antigen, the ratio of specific to unspecific phages was determined by analysing at least 91 independent infected cells via PCR. The PCR was performed according to standard protocols using single colonies as source of template and oligonucleotides specific for VH CDR3 and VL CDR3 of each scFv as primers. After 2 rounds of panning, ˜0% positive clones (0 out of 93 clones analysed) were obtained for the pMorph18-C-gIII-hag2-LHC panning and ˜3% positive clones (3 out of 91 clones analysed) were obtained for the pMorph18-C-gIII-MacI-5-LHC panning. After 3 rounds of panning, the specific clones were enriched to ˜79% (92 out of 117 clones analysed) for the pMorph18-C-gIII-hag2-LHC panning and to ˜100% (229 out of 229 clones analysed) for the pMorph18-C-gIII-MacI-5-LHC panning.

[0145] Enrichment of Engineered Phages Displaying scFv in Pannings of Pre-Selected Pools

[0146] To prove that engineered phages displaying scFvs can be selected out of a diverse pool, pannings of pre-selected libraries were performed. Pools after one round of conventional panning were subcloned into the engineered one-vector format and panning was continued for up to three further rounds (cys-display pannings).

[0147] Pannings were performed against the following antigens: (i) ICAMI comprising the extracellular part of mature ICAMI (amino acids 1-454) plus amino acids CGRDYKDDDKHHHHHH (SEQ ID NO:4) containing the M2-Flag and the 6x histidine tag. (ii) N1-MacI comprising aa 1-82 of mature gene III protein of phage M13 containing an additional methionine residue at the N-terminus plus a short linker at the C-terminus (N1), fused to a polypeptide containing amino acids 149-353 of human CR-3 alpha chain (SWISS-PROT entry P11215) plus the C-terminal sequence IEGRHHHHHH (SEQ ID NO:2) which includes the 6× histidine tag; and (iii) N1-Np50 comprising N1 fused to a polypeptide containing amino acids 2-366 of human NFKB p50 plus amino acids EFSHHHHHH (SEQ ID NO:5) which include the 6× histidine tag. Expression vectors for Ni-MacI and Ni-Np50 are based on vector pTFT74 (Freund et al., 1993) (complete vector sequence of pTFT74-N1-hag-HIPM given in FIG. 2). Expression, purification and refolding was done as described (Krebber, 1996; Krebber et al., 1997).

[0148] Initially, one round of conventional panning of the antibody library HuCAL-scFv (WO 97/08320; Knappik et al., 2000) was performed according to standard protocols. Briefly, wells of Maxisorp microtiterplates (Nunc; #442404) were coated with the respective antigen dissolved in PBS and blocked with 5 % skimmed milk powder in PBS. 1-5×10¹² HuCAL-scFv phage were added for 1 h at 20° C. After several washing steps with PBST (PBS, 0.05% Tween20) and PBS, bound phage were eluted either with 100 rnM triethylamine or 100 mM glycine pH 2.2, immediately neutralised with 1 M Tris/HCl pH 7.0 and used for infection of TG1 cells. In addition, residual phages were eluted by direct infection of TG1 cells added to the wells. Pannings against N1-Np50 used the complete HuCAL-scFv library (κ and λ pools combined), in pannings against N1-MacI κ and λ light chain pools were kept separated. Against ICAM1 one round of conventional panning of the λ light chain part of HuCAL-scFv was performed and subsequently the selected heavy chains again combined with the complete library of λ light chains. The resulting light chain optimised library had a diversity of 1.4×10⁷. The scFvs of the respective pools were subcloned into vector pMorph20-C-gIII-scFv-LHC (one-vector format) via the unique SphI and EcoRI sites. Subsequently, three rounds of cys-display panning were performed. Phages were prepared by standard procedure and pre-blocked by mixing 1:1 with PBSTM (PBS, 5% skimmed milk powder, 0.1% Tween20) and incubated for 2 hrs at RT. Wells of a Nunc Maxisorp plate (#442404) were coated with specific antigens at a concentration of 5 μg/well in PBS overnight at 4° C., and subsequently blocked with 400 μl PBSM (PBS, 5% skimmed milk powder) for 2 hrs at RT. For each round of cys-display panning, between 1×10¹⁰ and 4.5×10¹¹ pre-blocked phages were applied per well and incubated for 1 h at RT on a microtiter plate shaker. Phage solution was removed and wells were washed with PBST (PBS, 0.05% Tween20) and PBS with increasing stringency. The 1^(st) round was washed 3× quick and 2× 5 min with PBST and PBS, respectively, the 2^(nd) round 1× quick and 4× 5 min with PBST and PBS, respectively, and the 3^(rd) round 10× quick and 5×5 min with PBST and PBS, respectively. Bound phages were eluted with 100 mM triethylamine according to standard protocols and used for infection of TG1 cells. In addition, residual phages were eluted by direct infection of TG1 cells added to the wells.

[0149] After each round of panning the number of antigen specific phages was determined in an ELISA. N1-MacI, N1-Np50 and ICAM-Strep (comprising amino acids 1-455 of mature ICAM1 plus SAWSHPQFEK (SEQ ID NO:6) containing the Strep-tag II) were used as antigens, respectively. To ensure high level expression the selected scFvs were subcloned into expression vector pMorphX7-FS (Table 1). Subcloning was done in two steps. First the scFv fragments were isolated from pMorph20-C-gIII-scFv-LHC via AfII and EcoRI, then the fragments were re-digested with SphI and cloned into the EcoRI/SphI digested pMorphX7-FS vector. This procedure ensured that only scFvs from vector pMorph20-C-gIII-scFv-LHC were subcloned and excluded any contamination with scFvs from a conventional display or expression vector. Expression of the scFvs and their testing in ELISA against the respective antigens was done according to standard procedures. Clones which showed a signal of at least 3× above background in ELISA were considered positive. The results are summarised in Table 5. To prove that the selected scFvs bind strongly and specifically to their respective antigen several positive clones after 2 rounds of cys-display panning were selected and re-tested in quadruplicates in a specificity ELISA on six different antigens (FIGS. 14 & 15). Enrichment of antigen-specific binders could clearly be demonstrated. Already after two rounds of cys-display panning of the pre-selected pools against N1-MacI, N1-Np50 and ICAM1 between 80% and 97% of the tested clones were positive in ELISA. The affinity of some of the selected scFvs was determined in Biacore and Kd values in the range of 1 nM to 2.2 μM were determined. These results are similar to the enrichment factors and affinities obtained in a conventional panning of the respective pools performed in parallel. Some of the scFvs were selected independently via cys-display as well as conventional panning.

[0150] Elution of Engineered Phages Displaying scFv via Reducing Agents

[0151] When screening phage display libraries in biopanning the problem remains how to best recover phages which have bound to the desired target. Normally, this is achieved by elution with appropriate buffers, either by using a pH-or salt gradient, or by specific elution using soluble target. However, the most interesting binders which bind with high affinity to the target might be lost by that approach. One option with engineered cys-display phages is that the complexes of target and specific bacteriophages can be treated with reducing agents, e.g. by incubation with DTT, to cleave the disulfide bond between scFv and phage coat protein and to recover the specific bacteriophage particles.

[0152] Pannings of pre-selected pools against N1-MacI were performed according to the protocol described above. Phages were eluted either according to the standard protocol with 100 mM triethylamine and a direct infection of TG1 cells by residual phages, or by incubation of the wells with 20 mM DTT in Tris buffer pH 8.0 for 10 min. After each round of panning the pool of selected scFvs was subcloned into expression vector pMorphX7-FS according to the two step procedure described above, and the number of N1-MacI specific scFvs was determined in ELISA. To prove that the selected scFvs bind strongly and specifically to their respective antigen several positive clones were selected and re-tested in triplicates in a specificity ELISA. Enrichment of antigen-specific binders could clearly be demonstrated for both elution procedures. After two rounds of panning of the MacI K-pool and the MacI λ-pool a two-fold and five-fold, respectively, higher number of ELISA positive clones was obtained for elution with reducing agents compared to conventional elution.

EXAMPLE 2.2

[0153] Display of Fabs

[0154] Example 2.1 shows that functional single chain fragments can be displayed on engineered phages via disulfide bonds. In the following we will describe experiments which show that the same is true for Fabs. The cysteine was engineered at different positions of the Fab antibody fragment. These Fabs were co-expressed in the same bacterial cell together with engineered full length pIII based on a two-vector system.

[0155] Construction of Vectors Expressing Fabs and Engineered pIIIH

[0156] Heavy and light chains of the Fab fragment were expressed from a dicistronic phagemid under control of the lac promotor/operator region. The first expression cassette comprises the signal sequence ompA and the variable and constant domain of the light chain, the second expression cassette comprises the signal sequence phoA and the variable and constant domain of the heavy chain. Heavy and light chain are not linked via a disulfide bond. Modules containing the engineered cysteine were located at the C-terminus of either the light or the heavy chain. Several constructs differing in the amino acid composition of the modules were compared and are summarised in Table 6. As an example the complete vector sequence of pMorphX10-Fab-VL-LHC-VH-FS coding for the modified Fab MacI-5 and the respective vector map are given in FIG. 16a+b.

[0157] Two different plasmids were used for expression of full length pIlI. Plasmid pBR-C-gIII was already described above. The respective expression cassette comprises the signal sequence ompA, amino acids DYCDIEF (SEQ ID NO:3) and the pIII ORF under control of the lactose promotor/operator region (Table 3, FIG. 6). Alternatively, plasmid pBAD-SS-C-gIII was used. Here the respective expression cassette comprises the signal sequence of pIII, amino acids TMACDIEF (SEQ ID NO:7) and the pIII ORF under control of the arabinose promotor/operator region (Table 3). For construction of pBAD-SS-C-gIII the fragment coding for the engineered cysteine plus pIII was amplified from pUC-C-gIII via PCR introducing the restriction sites NcoI and HindIII and cloned into the commercially available vector pBAD/gIII A (Invitrogen). The plasmids pBR-C-gIII or pBAD-SS-C-gIII were co-transformed with the respective pMorphX10-Fab phagemids expressing the modified Fab into E.coli TG1 selecting for both antibiotic markers.

[0158] Description of the Fab-Antigen Interactions

[0159] Three different Fabs all deriving from a human combinatorial antibody library (HuCAL; WO 97/08320; Knappik et al., 2000) were used for evaluation of Fab display on engineered phage. The HuCAL VH and VL consensus genes (described in WO 97/08320), and the CDR3 sequences of the Fabs are given in Table 2. Fab MacI-5 is derived from the scFv MacI-5 described above and was converted into the Fab format (complete vector map of pMorphX10-Fab-MacI5-VL-LHC-VH-FS is given in FIG. 16a). Fabs MacI-A8 and ICAM1-C8 were isolated directly from one of the HuCAL-Fab libraries. Clone MacI-A8 was selected against antigen MacI-Strep, which comprises an N-terminal methionine, amino acids 149-353 of human CR-3 alpha chain (SWISS-PROT entry P11215) and amino acids SAWSHPQFEK (SEQ ID NO:6) which include the Strep-tag II (Schmidt et al., 1996). Expression and purification were done according to Schmidt & Skerra (1994). N1-MacI was used as corresponding antigen for ELISAs. N1-MacI is described above, and comprises an N-terminal methionine, amino acids 1-82 of mature gene III protein of phage M13 plus a short linker (N1), amino acids 149-353 of human CR-3 alpha chain (SWISS-PROT entry P11215) and amino acids IEGRHHHHHH (SEQ ID NO:2) which include the 6× histidine tag. Clone ICAM1-C8 was selected against antigen ICAM1 described above, which comprises the extracellular part of mature ICAM1 (amino acids 1-454) plus amino acids CGRDYKDDDKHHHHHH (SEQ ID NO:4) containing the M2-Flag and the 6× histidine tags. The same antigen was used for ELISA assays as well as in the doped library experiment.

[0160] Attachment of Fabs to Phage Coat Proteins Via Disulfide Bonds

[0161] To demonstrate that the Fabs attach to pIII via disulfide bridges and are incorporated into phage particles, the respective phages were run on SDS PAGE under non-reducing and reducing conditions. Western blot analyses were performed with antibodies detecting pIII, the heavy chain, the lambda light and the kappa light chain, respectively. All constructs described in table 6 were analysed and the results are shown for pMorphX10-ICAM1C8-VL-LHC-VH-FS plus pBAD-SS-C-gIII and pMorphX10-MacIA8-VL-LHC-VH-MS plus pBAD-SS-C-gIII as an example in FIGS. 17 and 18.

[0162] Phages were produced using helper phage VCSM13 following standard protocols (Kay et al., 1996). In addition to helper phage proteins, engineered phage coat protein and soluble modified Fab were co-expressed from the two-vector system. Phages were pre-incubated in PBS with or without 20 mM DTT (reducing and non-reducing conditions, respectively) for 1 h at room temperature before adding SDS loading buffer lacking reducing agents such as DTT or β-mercaptoethanol. 1×10¹⁰ phages per lane were run on a 12 % SDS PAGE (BioRad) and blotted onto nitrocellulose membranes (Schleicher & Schuell). For the anti-pIII Western blot, the membrane was blocked in MTBST (50 mM Tris buffer pH 7.4, containing 5% milk powder and 0.05% Tween20) and developed with mouse anti-pIII (1:250 dilution, Mobitec) as primary antibody, anti-mouse-IgG-HRP conjugate (1:5000 dilution; SIGMA) as secondary antibody and BM Blue POD precipitating (Roche #1442066) as substrate. For the detection of the heavy chain, kappa light and lambda light chain the primary antibodies anti-Fd (1:5000 dilution; The binding site PC075), anti-human kappa (1:5000 dilution; Sigma K-4377) and anti-human lambda (1:500 dilution, Sigma L-6522) were used, respectively.

[0163] In the anti-pIII Western blots, free protein III (SH-pIII and/or pIII) can be detected for all expression systems both under reducing and non-reducing conditions (FIGS. 17 and 18). When both engineered Fab and engineered protein III are co-expressed a signal migrating at the height of a hetero-dimer of light chain and protein III (VL-CL-SS-pIII) appears under non-reducing conditions. In addition, a band migrating at the height expected for disulfide bonded protein III dimers (pIII-SS-pIII) can be seen (lanes 11 & 12, FIGS. 17, 18). Both hetero-and homo-dimers disappear when the samples are treated with DTT (lanes 5 & 6, FIGS. 17 and 18) or when modified Fabs are coexpressed with non-engineered pIII (lanes 3, 4, 9 & 10, FIGS. 17 and 18). The hetero-dimer in this case of light chain linked to the full length pIII could also be detected with anti-light chain antibodies in non-reducing gels but was absent under reducing conditions. In addition, a band migrating at the height expected for the homo-dimer of the light chain (VL-CL-SS-VL-CL) was detectable (data not shown). Similar results were obtained for all constructs described in Table 6, and no significant difference between vectors pBR-C-gIII and pBAD-SS-C-gIII for supply of engineered pIII was detected (data not shown).

[0164] Functionality of Fabs Displayed on Engineered Phages

[0165] To show that the displayed Fabs are functional with respect to recognition of the specific antigen phage ELISAs were performed. The analysis was done for the HuCAL Fabs MacI-5, MacI-A8 and ICAM1-C8. All formats differing in the position of cysteine at the Fab were compared (Table 6). To demonstrate that the Fabs attach to the engineered phage coat proteins via disulfide bonds, phage ELISAs were performed both under non-reducing and reducing conditions.

[0166] The respective phagemids expressing the modified Fab were co-transformed with pBR-C-gIII and phage production was performed under standard conditions (Kay et al., 1996). Conventional Fab display phages (pMorph18-Fab) served as positive control, a phagemid expression vector for expression of non-engineered Fab (pMorphX9-Fab-FS) served as negative control. Specific antigen or control antigen (BSA, Sigma #A7906) was coated for 12 hours at 4° C. at an amount of 5 μg/well in PBS to Nunc Maxisorp microtiter plates (# 442404) and blocked with PBS containing 5% skimmed milk powder, 0.05% Tween 20 for I h. Phages were pre-incubated in PBS containing 5% skimmed milk powder, 0.05% Tween 20, and 10 mM DTT where applicable for 1 h at room temperature before they were applied to the ELISA well coated with antigen at a concentration range between 1×10⁸ and 1×10¹⁰ phages per well. After binding for 1 h at RT, unspecifically bound phages were washed away with PBS containing 0.05% Tween 20 and PBS. Bound phages were detected in ELISA using an anti-M13-HRP conjugate (Amersham Pharmacia Biotech #27-9421-01) and BM blue soluble (Roche #1484281). Absorbance at 370 nm was measured. ELISA signals obtained with the specific antigen were compared to those with the control. Up to three independent phage preparations were analysed and mean values are given in FIGS. 19 to 22.

[0167] For all different two-vector formats specific binding of Fab displaying phages to antigen could be demonstrated (FIGS. 19-21, lanes 1-4). For Fab MacI-5 no significant difference between the four formats was detected (FIG. 19), while construct pMorphX10-Fab-VL-LHC-VH-FS showed reproducibly best results for Fab MacI-A8 and ICAM1-C8 (FIGS. 20 and 21). When 10 mM DTT was added to the phages prior to antigen binding during the pre-incubation step, the ELISA signal was decreased to almost background levels for all cys-display phages while DTT had no major effect on conventional display phages (pMorphl8-Fab) (shown for Fab MacI-5 in FIG. 22). This shows that disulfide bonds are essential for the functional display of Fabs on phages and thus for the specific binding of Fab displaying phages to antigen.

[0168] Enrichment of Engineered Phages Displaying Fabs in Doped Library Experiments

[0169] To prove that engineered phages displaying Fabs can be enriched on specific antigen, a “doped library” experiment was performed: specific phages were mixed with a high excess of unspecific phages and three rounds of panning on specific antigen were performed. The enrichment for specific phages was determined after each round. The analysis was done for the HuCAL Fab ICAM1-C8 in the two vector system pMorphXI10-Fab-VL-LHC-VH-FS plus pBAD-SS-C-gIII.

[0170] Engineered phages displaying ICAM1-C8 and MacI-A8 were mixed at ratios of 1:10⁵. Three rounds of panning were performed on the ICAM1 antigen. Phages were prepared by standard procedure, pre-blocked by mixing 1:1 with PBSTM (PBS, 5% skimmed milk powder, 0.1% Tween 20) and incubated for 2 hrs at RT. Wells of a Nunc Maxisorp plate (#442404) were coated with specific antigen at a concentration of 5 μg/well in PBS overnight at 4° C., and subsequently blocked with 400 μl PBSM (PBS, 5% skimmed milk powder) for 2 hrs at RT. For the first round, 10¹¹ pre-blocked phages were applied per well and incubated for 1 h at RT on a microtiter plate shaker. Phage solution was removed and wells were washed 3 times with PBST (PBS, 0.05% Tween 20; 1× quick, 2× 5 min) and 3 times with PBS (1× quick, 2×5 min). Bound phages were eluted with 100 mM triethylamine according to standard protocols. In addition, residual phages were eluted by direct infection of cells added to the wells. As a direct infection of TG1 cells harbouring pBAD-SS-C-gIII was not efficient enough, eluted phages were used for infection of TG1 cells, amplified and than used for infection of TG1 cells harbouring pBAD-SS-C-gIII. Thus the two-vector system was restored and the next round of panning was performed. While no difference between the two plasmids for expression of engineered pll (pBR-C-gIII and pBAD-SS-C-gIII) was observed with respect to phage ELISA and WB, infection of TG1 cells harbouring pBR-C-gIII was not as efficient as infection of TG1 cells harbouring pBAD-SS-C-gIII. After each round of panning the ratio of specific to unspecific phages was determined by analysing at least 92 independent infected cells via PCR. The PCR was performed according to standard protocols using single colonies as source of template and oligonucleotides specific for the lambda light chain (priming in framework 4), the kappa light chain (priming in framework 3) and a vector sequence upstream of the Fab fragment (commercial M13-rev primer,NEB) as primers. Fragments of roughly 420 bp length were expected for lambda Fabs (ICAM1-C8) and 290 bp for kappa Fabs (MacI-A8). After 2 rounds of panning, 61% positive clones (57 out of 93 clones analysed) were obtained, which could be enriched to 100% (92 out of 92 clones analysed) after the third round.

[0171] References

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[0218] TABLE 2 Amino acid sequence of HuCAL scFvs and HuCAL Fabs* scFv antigen VH VH CDR3 VL VL CDR3 hag2 peptide of influenza virus VH3 RSGAYDY VK4 QQYSSFPL hemagglutinine (SEQ ID NO. (SEQ ID NO: (CAGPYDVPDYASLRSHH) (SEQ ID NO: AB 1.1 12 amino acid peptide VH3 10 amino acid Vλ1 9 amino acid residues residues MacI-5 fragment of human CR-3 VH2 FDPFFDSFFDY Vλ1 QSYDQNALVE alpha chain (SEQ ID NO:17) (SEQ ID NO:18) MacI-A8 fragment of human CR-3 VH3 HGYRKYYTDM Vλ1 HQVYSTSP alpha chain FDV (SEQ ID NO:20) (SEQ ID NO:19) ICAM1-C8 human ICAM1 VH2 FPYTYTIGFMID Vλ3 QSYDSGNL N (SEQ ID NO:22) (SEQ ID NO:21)

[0219] TABLE 3 Amino acid sequence of engineered phage coat proteins of vector pBR-C-gIII and derivatives EcoRV- Construct Signal Sequence EcoRI sequence HindIII pUC-C-gIII MKKTAIAIAVAL DYC DI EF AETVESCLAKPHTENSFTNYWKDD stop pBR-C-gIII AGFATVAQA KTLDRYANYEGCLWNATGVVVCT (ompA) GDETOCYGTWVPJGLAIPENEGGGS EGGGSEGGGSEGGGTKPPEYGDTPI PGYTYPNPLDGTYPPGTEQNPAMN PSLEESOPLNTFMFQThRfRNRQGA LTVYTGTVTOGTDPVKTYYOYTPV SSKAMYDAYWNGKFRDCAFHSGF NEDPFVCEYOGQSSDLPOPPVNAG GGSGGGSGGGSEGGGSEGGGSEGG GSEGGGSGGGSGSGDFDYEKMAN ANKGAMTENADENALOSDAKGKL DSVATDYGAAIDGFIGDVSGLANG NGATGDFAGSNSQMAQVGDGDNS PLMNNFROYLPSLPQSVECRPYVFG AGKPYEFSIDCDKTNLFRGVFAFLLY VATFMYVFSTFAMILRLNKES (SEQ ID NO:23) pUC-C- MKKTAIAIAVAL DYC DI EF NAGGGSGGGSGGGSEGGGSEGGGS stop gIIICT AGFATVAQA EGGGSEGGGSGGGSGSGDFDYEK pBR-C- (ompA) MANANKGAMTENADENALOSDAK gIIICT GKLDSVATDYGAAIDGFIGDVSGL ANGNGATGDFAGSNSOMAOVGDG DNSPLMNNFRQYLPSLPOSVECRPF VFGAGKPYEFSTDCDKINLFRGVFA FLLYVATFMYVFSTFANILRNKES (SEQ ID NO:24) pBAD-SS- MIKKLLFAIPLVVTMA DY DI EF AETVESCLAKPHTENSFTNVWKDDKT stop C-gIII PFYSHS C LDRYANYEGCLWNATGVVVCTGDET (gIII) NcoI QCYGTWVPIGLAIPENEGGGSEGGGSE (StyI) GGGSEGGGTKPPEYGDTPIPGYTYINP /SphI LDGTYPPGTEQNPANPNPSLEESQPLN TFMFQNNRFRNRQGALTVYTGTVTQG TDPVKTYYQYTPVSSKAMYDAYWNG KFRDCAFHSGFNEDPFVCEYQGQSSDL PQPPVNAGGGSGGGSGGGSEGGGSEG GGSEGGGSEGGGSGGGSGSGDFDYEK MANANKGAMTENADENALQSDAKGK LDSVATDYGAAIDGFIGDVSGLANGNG ATGDFAGSNSQMAQVGDGDNSPLMN NFRQYLPSLPQSVECRYVFGAGKPYE FSIDCDKJNLFRGVFAFLLYVATFMYV FSTFANILRNKES (SEQ ID NO:25)

[0220] TABLE 4 Amino acid sequence of engineered phage coat proteins of vector pMorph18- C-gIII-scFv-LHC and derivatives OmpA EcoRV Construct Signal Sequence EcoRI sequence StuI pMorph18-C- MKKTAIAIAVAL DY DI EF AETVESCLAKPHTENSFTNVSKD stop gIII-scFv- AGFATVAQA C DKTLDRYANYEGCLWNATGVVV LHC CTGDETQCYGTWVPIGLAIPENEG GGSEGGGSEGGGSEGGGTKPPEY GDTPIPGYTYIMNPLDGTYPPGTEQ NPANPNPSLEESOPLNTFMQNNR FRNRQGALTVYTGTVTOGTDPVK TYYOYTPVSSKAMYDAYWNGKF RDCAFHSGFNEDPFVCEYOGQSSD LPQPPVNAGGGSGGGSGGGSEEG GSEGGGSEGGGSEGGGSGGGSGS GDFDYEKMANANKGAMTENADE NALOSDAKGKLDSVATDYGAAID GFIGDVSGLANGNGATGDFAGSN SQMAOVGDGDNSPLMINMBRQYL PSLPQSVECRPYVFGAGKYYEFSID CDKTNLFRGVFAFLLYVATFMYXTF STFANILRNKES (SEQ lID NO:26) pMorph18-C- MKKTAIAIAVAL DY DI EF NAGGGSGGGSGSEGGGSEGGG stop gIIICT-scFv- AGFATVAQA C SEGGGSEGGGSGGGSGSGDFDYE LHC KMANANKGAMTENADENAIOSD AKGKLD SVATDYGAAIDGFIGDVS GLANGNGATGDFAGSNSQMAOV GDGDNSPLMNNFRQYLPSLPQSV ECRPFVFGAGKPYEFSIDCDKINLF RGVFAFLLYVATFMYVFSTFANIL RINKES (SEQ ID NO:27) pMorph18-C- MIKKTAIAIAVAL DY DI EF GGGGSMSVLVYSFASFVLGWCLR stop gIX-scFv- AGFATVAQA C SGITYFTRLMETSS LHC (SEQ ID NO:28)

[0221] TABLE 5 Cys-display panning of pre-selected pools Preselected # of # of Panning Pool clones^(a) positives^(b) Format round 1 round 2 round 3 N1-MacI   2 × 10⁵ 3/186 = Cys-display 78/279 = 28% 89/93 = 96% 92/93 = 99% κ chains 2% conventional 10/93 = 11% 71/93 = 76% nd N1-MacI   4 × 10⁴ 4/186 = Cys-display 72/279 = 26% 90/93 = 97% 90/93 = 97% λ chains 2% conventional 34/93 = 37% 87/93 = 94% nd N1-Np50   5 × 10⁴ 0/186 = Cys-display 17/93 = 18% 244/279 = 87% nd 0% conventional 51/93 = 55% 86/93 = 92% nd ICAM1 1.4 × 10⁷ nd Cys-display 4/186 = 2% 149/186 = 80% nd

[0222] TABLE 6 Amino acid sequence of modules of engineered Fab fragment Module at the light chain Module at the heavy chain Construct elements amino acids elements amino acids pMorphX10-Fab- linker- SPGGSG-GAP- linker EF- VL-LHC-VH-FS histidine tag- HHHHHH- Flag tag-linker DYKDDDDK-GAP- cysteine C-stop Strep-tag II WSLIPQFEK-stop (SEQ ID NO:29) (SEQ ID NO:30) pMorphX10-Fab- linker- SPGGSG-GAP- linker- EF- VL-LHC-VH-MS histidine tag- HHHHHH- myc tag-linker- EQKLISEEDLN-GAP- cysteine C-stop Strep-tag II WSHIPQFEK-stop (SEQ ID NO:29) (SEQ ID NO:31) pMorphX10-Fab- cysteine deletion of A- linker- EF- VL-C-VH-FS C-stop (κ-chains) Flag tag-linker- DYKDDDDK-GAP- CS-stop (λ-chains) Strep-tag II WSIIPQFEK-stop (SEQ ID NO:30) pMorphX10-Fab- cysteine deletion of A- linker- EF- VL-C-VH-MS C-stop (κ-chains) myc tag-linker- EQKLISEEDLN-GAP- CS-stop (λ-chains) Strep-tag II WSFEPQFEK-stop (SEQ ID NO:3 1) pMorphX10-Fab- — — linker- EF-PGGSG-GAP- VL-VH-LHC histidine tag- HHHHHH- cysteine C-stop (SEQ ID NO:32) pMorphX10-Fab- — — cysteine-linker- C-EF- VL-VH-CFS Flag tag-linker- DYKDDDDK-GAP- Strep-tag II WSHPQFEK-stop (SEQ ID NO:33) pMorphX10-Fab- — — cysteine-linker- C-EF- VL-VH-CMS myc tag-linker- EQKLISEEDLN-GAP- Strep-tag II WSHPQFEK-stop (SEQ ID NO:34)

[0223]

1 41 1 18 PRT artificial sequence Description of Artificial Sequence synthetic module 1 Pro Tyr Asp Val Pro Asp Tyr Ala Ser Leu Arg Ser His His His His 1 5 10 15 His His 2 10 PRT artificial sequence Description of Artificial Sequence synthetic module 2 Ile Glu Gly Arg His His His His His His 1 5 10 3 7 PRT artificial sequence Description of Artificial Sequence synthetic module 3 Asp Tyr Cys Asp Ile Glu Phe 1 5 4 16 PRT artificial sequence Description of Artificial Sequence synthetic module 4 Cys Gly Arg Asp Tyr Lys Asp Asp Asp Lys His His His His His His 1 5 10 15 5 9 PRT artificial sequence Description of Artificial Sequence synthetic module 5 Glu Phe Ser His His His His His His 1 5 6 10 PRT artificial sequence Description of Artificial Sequence synthetic module 6 Ser Ala Trp Ser His Pro Gln Phe Glu Lys 1 5 10 7 8 PRT artificial sequence Description of Artificial Sequence synthetic module 7 Thr Met Ala Cys Asp Ile Glu Phe 1 5 8 8 PRT artificial sequence Description of Artificial Sequence synthetic module 8 Asp Tyr Lys Asp Asp Asp Asp Lys 1 5 9 8 PRT artificial sequence Description of Artificial Sequence synthetic module 9 Trp Ser His Pro Gln Phe Glu Lys 1 5 10 5 PRT artificial sequence Description of Artificial Sequence synthetic module 10 Pro Gly Gly Ser Gly 1 5 11 6 PRT artificial sequence Description of Artificial Sequence synthetic module 11 His His His His His His 1 5 12 7 PRT artificial sequence Description of Artificial Sequence synthetic module 12 Cys His His His His His His 1 5 13 7 PRT artificial sequence Description of Artificial Sequence synthetic module 13 His His His His His His Cys 1 5 14 17 PRT artificial sequence Description of Artificial Sequence synthetic module 14 Cys Ala Gly Pro Tyr Asp Val Pro Asp Tyr Ala Ser Leu Arg Ser His 1 5 10 15 His 15 7 PRT artificial sequence Description of Artificial Sequence synthetic module 15 Arg Ser Gly Ala Tyr Asp Tyr 1 5 16 8 PRT artificial sequence Description of Artificial Sequence synthetic module 16 Gln Gln Tyr Ser Ser Phe Pro Leu 1 5 17 11 PRT artificial sequence Description of Artificial Sequence synthetic module 17 Phe Asp Pro Phe Phe Asp Ser Phe Phe Asp Tyr 1 5 10 18 10 PRT artificial sequence Description of Artificial Sequence synthetic module 18 Gln Ser Tyr Asp Gln Asn Ala Leu Val Glu 1 5 10 19 13 PRT artificial sequence Description of Artificial Sequence synthetic module 19 His Gly Tyr Arg Lys Tyr Tyr Thr Asp Met Phe Asp Val 1 5 10 20 8 PRT artificial sequence Description of Artificial Sequence synthetic module 20 His Gln Val Tyr Ser Thr Ser Pro 1 5 21 11 PRT artificial sequence Description of Artificial Sequence synthetic module 21 Phe Pro Tyr Thr Tyr His Gly Phe Met Asp Asn 1 5 10 22 8 PRT artificial sequence Description of Artificial Sequence synthetic module 22 Gln Ser Tyr Asp Ser Gly Asn Leu 1 5 23 434 PRT artificial sequence Description of Artificial Sequence synthetic module 23 Met Lys Lys Thr Ala Ile Ala Ile Ala Val Ala Leu Ala Gly Phe Ala 1 5 10 15 Thr Val Ala Gln Ala Asp Tyr Cys Asp Ile Glu Phe Ala Glu Thr Val 20 25 30 Glu Ser Cys Leu Ala Lys Pro His Thr Glu Asn Ser Phe Thr Asn Val 35 40 45 Trp Lys Asp Asp Lys Thr Leu Asp Arg Tyr Ala Asn Tyr Glu Gly Cys 50 55 60 Leu Trp Asn Ala Thr Gly Val Val Val Cys Thr Gly Asp Glu Thr Gln 65 70 75 80 Cys Tyr Gly Thr Trp Val Pro Ile Gly Leu Ala Ile Pro Glu Asn Glu 85 90 95 Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly 100 105 110 Gly Gly Thr Lys Pro Pro Glu Tyr Gly Asp Thr Pro Ile Pro Gly Tyr 115 120 125 Thr Tyr Ile Asn Pro Leu Asp Gly Thr Tyr Pro Pro Gly Thr Glu Gln 130 135 140 Asn Pro Ala Asn Pro Asn Pro Ser Leu Glu Glu Ser Gln Pro Leu Asn 145 150 155 160 Thr Phe Met Phe Gln Asn Asn Arg Phe Arg Asn Arg Gln Gly Ala Leu 165 170 175 Thr Val Tyr Thr Gly Thr Val Thr Gln Gly Thr Asp Pro Val Lys Thr 180 185 190 Tyr Tyr Gln Tyr Thr Pro Val Ser Ser Lys Ala Met Tyr Asp Ala Tyr 195 200 205 Trp Asn Gly Lys Phe Arg Asp Cys Ala Phe His Ser Gly Phe Asn Glu 210 215 220 Asp Pro Phe Val Cys Glu Tyr Gln Gly Gln Ser Ser Asp Leu Pro Gln 225 230 235 240 Pro Pro Val Asn Ala Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly 245 250 255 Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser 260 265 270 Glu Gly Gly Gly Ser Gly Gly Gly Ser Gly Ser Gly Asp Phe Asp Tyr 275 280 285 Glu Lys Met Ala Asn Ala Asn Lys Gly Ala Met Thr Glu Asn Ala Asp 290 295 300 Glu Asn Ala Leu Gln Ser Asp Ala Lys Gly Lys Leu Asp Ser Val Ala 305 310 315 320 Thr Asp Tyr Gly Ala Ala Ile Asp Gly Phe Ile Gly Asp Val Ser Gly 325 330 335 Leu Ala Asn Gly Asn Gly Ala Thr Gly Asp Phe Ala Gly Ser Asn Ser 340 345 350 Gln Met Ala Gln Val Gly Asp Gly Asp Asn Ser Pro Leu Met Asn Asn 355 360 365 Phe Arg Gln Tyr Leu Pro Ser Leu Pro Gln Ser Val Glu Cys Arg Pro 370 375 380 Tyr Val Phe Gly Ala Gly Lys Pro Tyr Glu Phe Ser Ile Asp Cys Asp 385 390 395 400 Lys Ile Asn Leu Phe Arg Gly Val Phe Ala Phe Leu Leu Tyr Val Ala 405 410 415 Thr Phe Met Tyr Val Phe Ser Thr Phe Ala Asn Ile Leu Arg Asn Lys 420 425 430 Glu Ser 24 219 PRT artificial sequence Description of Artificial Sequence synthetic module 24 Met Lys Lys Thr Ala Ile Ala Ile Ala Val Ala Leu Ala Gly Phe Ala 1 5 10 15 Thr Val Ala Gln Ala Asp Tyr Cys Asp Ile Glu Phe Asn Ala Gly Gly 20 25 30 Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu 35 40 45 Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Gly Gly 50 55 60 Gly Ser Gly Ser Gly Asp Phe Asp Tyr Glu Lys Met Ala Asn Ala Asn 65 70 75 80 Lys Gly Ala Met Thr Glu Asn Ala Asp Glu Asn Ala Leu Gln Ser Asp 85 90 95 Ala Lys Gly Lys Leu Asp Ser Val Ala Thr Asp Tyr Gly Ala Ala Ile 100 105 110 Asp Gly Phe Ile Gly Asp Val Ser Gly Leu Ala Asn Gly Asn Gly Ala 115 120 125 Thr Gly Asp Phe Ala Gly Ser Asn Ser Gln Met Ala Gln Val Gly Asp 130 135 140 Gly Asp Asn Ser Pro Leu Met Asn Asn Phe Arg Gln Tyr Leu Pro Ser 145 150 155 160 Leu Pro Gln Ser Val Glu Cys Arg Pro Phe Val Phe Gly Ala Gly Lys 165 170 175 Pro Tyr Glu Phe Ser Ile Asp Cys Asp Lys Ile Asn Leu Phe Arg Gly 180 185 190 Val Phe Ala Phe Leu Leu Tyr Val Ala Thr Phe Met Tyr Val Phe Ser 195 200 205 Thr Phe Ala Asn Ile Leu Arg Asn Lys Glu Ser 210 215 25 432 PRT artificial sequence Description of Artificial Sequence synthetic module 25 Met Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ser 1 5 10 15 His Ser Thr Met Ala Cys Asp Ile Glu Phe Ala Glu Thr Val Glu Ser 20 25 30 Cys Leu Ala Lys Pro His Thr Glu Asn Ser Phe Thr Asn Val Trp Lys 35 40 45 Asp Asp Lys Thr Leu Asp Arg Tyr Ala Asn Tyr Glu Gly Cys Leu Trp 50 55 60 Asn Ala Thr Gly Val Val Val Cys Thr Gly Asp Glu Thr Gln Cys Tyr 65 70 75 80 Gly Thr Trp Val Pro Ile Gly Leu Ala Ile Pro Glu Asn Glu Gly Gly 85 90 95 Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly 100 105 110 Thr Lys Pro Pro Glu Tyr Gly Asp Thr Pro Ile Pro Gly Tyr Thr Tyr 115 120 125 Ile Asn Pro Leu Asp Gly Thr Tyr Pro Pro Gly Thr Glu Gln Asn Pro 130 135 140 Ala Asn Pro Asn Pro Ser Leu Glu Glu Ser Gln Pro Leu Asn Thr Phe 145 150 155 160 Met Phe Gln Asn Asn Arg Phe Arg Asn Arg Gln Gly Ala Leu Thr Val 165 170 175 Tyr Thr Gly Thr Val Thr Gln Gly Thr Asp Pro Val Lys Thr Tyr Tyr 180 185 190 Gln Tyr Thr Pro Val Ser Ser Lys Ala Met Tyr Asp Ala Tyr Trp Asn 195 200 205 Gly Lys Phe Arg Asp Cys Ala Phe His Ser Gly Phe Asn Glu Asp Pro 210 215 220 Phe Val Cys Glu Tyr Gln Gly Gln Ser Ser Asp Leu Pro Gln Pro Pro 225 230 235 240 Val Asn Ala Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Glu 245 250 255 Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly 260 265 270 Gly Gly Ser Gly Gly Gly Ser Gly Ser Gly Asp Phe Asp Tyr Glu Lys 275 280 285 Met Ala Asn Ala Asn Lys Gly Ala Met Thr Glu Asn Ala Asp Glu Asn 290 295 300 Ala Leu Gln Ser Asp Ala Lys Gly Lys Leu Asp Ser Val Ala Thr Asp 305 310 315 320 Tyr Gly Ala Ala Ile Asp Gly Phe Ile Gly Asp Val Ser Gly Leu Ala 325 330 335 Asn Gly Asn Gly Ala Thr Gly Asp Phe Ala Gly Ser Asn Ser Gln Met 340 345 350 Ala Gln Val Gly Asp Gly Asp Asn Ser Pro Leu Met Asn Asn Phe Arg 355 360 365 Gln Tyr Leu Pro Ser Leu Pro Gln Ser Val Glu Cys Arg Pro Tyr Val 370 375 380 Phe Gly Ala Gly Lys Pro Tyr Glu Phe Ser Ile Asp Cys Asp Lys Ile 385 390 395 400 Asn Leu Phe Arg Gly Val Phe Ala Phe Leu Leu Tyr Val Ala Thr Phe 405 410 415 Met Tyr Val Phe Ser Thr Phe Ala Asn Ile Leu Arg Asn Lys Glu Ser 420 425 430 26 434 PRT artificial sequence Description of Artificial Sequence synthetic module 26 Met Lys Lys Thr Ala Ile Ala Ile Ala Val Ala Leu Ala Gly Phe Ala 1 5 10 15 Thr Val Ala Gln Ala Asp Tyr Cys Asp Ile Glu Phe Ala Glu Thr Val 20 25 30 Glu Ser Cys Leu Ala Lys Pro His Thr Glu Asn Ser Phe Thr Asn Val 35 40 45 Trp Lys Asp Asp Lys Thr Leu Asp Arg Tyr Ala Asn Tyr Glu Gly Cys 50 55 60 Leu Trp Asn Ala Thr Gly Val Val Val Cys Thr Gly Asp Glu Thr Gln 65 70 75 80 Cys Tyr Gly Thr Trp Val Pro Ile Gly Leu Ala Ile Pro Glu Asn Glu 85 90 95 Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly 100 105 110 Gly Gly Thr Lys Pro Pro Glu Tyr Gly Asp Thr Pro Ile Pro Gly Tyr 115 120 125 Thr Tyr Ile Asn Pro Leu Asp Gly Thr Tyr Pro Pro Gly Thr Glu Gln 130 135 140 Asn Pro Ala Asn Pro Asn Pro Ser Leu Glu Glu Ser Gln Pro Leu Asn 145 150 155 160 Thr Phe Met Phe Gln Asn Asn Arg Phe Arg Asn Arg Gln Gly Ala Leu 165 170 175 Thr Val Tyr Thr Gly Thr Val Thr Gln Gly Thr Asp Pro Val Lys Thr 180 185 190 Tyr Tyr Gln Tyr Thr Pro Val Ser Ser Lys Ala Met Tyr Asp Ala Tyr 195 200 205 Trp Asn Gly Lys Phe Arg Asp Cys Ala Phe His Ser Gly Phe Asn Glu 210 215 220 Asp Pro Phe Val Cys Glu Tyr Gln Gly Gln Ser Ser Asp Leu Pro Gln 225 230 235 240 Pro Pro Val Asn Ala Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly 245 250 255 Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser 260 265 270 Glu Gly Gly Gly Ser Gly Gly Gly Ser Gly Ser Gly Asp Phe Asp Tyr 275 280 285 Glu Lys Met Ala Asn Ala Asn Lys Gly Ala Met Thr Glu Asn Ala Asp 290 295 300 Glu Asn Ala Leu Gln Ser Asp Ala Lys Gly Lys Leu Asp Ser Val Ala 305 310 315 320 Thr Asp Tyr Gly Ala Ala Ile Asp Gly Phe Ile Gly Asp Val Ser Gly 325 330 335 Leu Ala Asn Gly Asn Gly Ala Thr Gly Asp Phe Ala Gly Ser Asn Ser 340 345 350 Gln Met Ala Gln Val Gly Asp Gly Asp Asn Ser Pro Leu Met Asn Asn 355 360 365 Phe Arg Gln Tyr Leu Pro Ser Leu Pro Gln Ser Val Glu Cys Arg Pro 370 375 380 Tyr Val Phe Gly Ala Gly Lys Pro Tyr Glu Phe Ser Ile Asp Cys Asp 385 390 395 400 Lys Ile Asn Leu Phe Arg Gly Val Phe Ala Phe Leu Leu Tyr Val Ala 405 410 415 Thr Phe Met Tyr Val Phe Ser Thr Phe Ala Asn Ile Leu Arg Asn Lys 420 425 430 Glu Ser 27 219 PRT artificial sequence Description of Artificial Sequence synthetic module 27 Met Lys Lys Thr Ala Ile Ala Ile Ala Val Ala Leu Ala Gly Phe Ala 1 5 10 15 Thr Val Ala Gln Ala Asp Tyr Cys Asp Ile Glu Phe Asn Ala Gly Gly 20 25 30 Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu 35 40 45 Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Gly Gly 50 55 60 Gly Ser Gly Ser Gly Asp Phe Asp Tyr Glu Lys Met Ala Asn Ala Asn 65 70 75 80 Lys Gly Ala Met Thr Glu Asn Ala Asp Glu Asn Ala Leu Gln Ser Asp 85 90 95 Ala Lys Gly Lys Leu Asp Ser Val Ala Thr Asp Tyr Gly Ala Ala Ile 100 105 110 Asp Gly Phe Ile Gly Asp Val Ser Gly Leu Ala Asn Gly Asn Gly Ala 115 120 125 Thr Gly Asp Phe Ala Gly Ser Asn Ser Gln Met Ala Gln Val Gly Asp 130 135 140 Gly Asp Asn Ser Pro Leu Met Asn Asn Phe Arg Gln Tyr Leu Pro Ser 145 150 155 160 Leu Pro Gln Ser Val Glu Cys Arg Pro Phe Val Phe Gly Ala Gly Lys 165 170 175 Pro Tyr Glu Phe Ser Ile Asp Cys Asp Lys Ile Asn Leu Phe Arg Gly 180 185 190 Val Phe Ala Phe Leu Leu Tyr Val Ala Thr Phe Met Tyr Val Phe Ser 195 200 205 Thr Phe Ala Asn Ile Leu Arg Asn Lys Glu Ser 210 215 28 65 PRT artificial sequence Description of Artificial Sequence synthetic module 28 Met Lys Lys Thr Ala Ile Ala Ile Ala Val Ala Leu Ala Gly Phe Ala 1 5 10 15 Thr Val Ala Gln Ala Asp Tyr Cys Asp Ile Glu Phe Gly Gly Gly Gly 20 25 30 Ser Met Ser Val Leu Val Tyr Ser Phe Ala Ser Phe Val Leu Gly Trp 35 40 45 Cys Leu Arg Ser Gly Ile Thr Tyr Phe Thr Arg Leu Met Glu Thr Ser 50 55 60 Ser 65 29 16 PRT artificial sequence Description of Artificial Sequence synthetic module 29 Ser Pro Gly Gly Ser Gly Gly Ala Pro His His His His His His Cys 1 5 10 15 30 21 PRT artificial sequence Description of Artificial Sequence synthetic module 30 Glu Phe Asp Tyr Lys Asp Asp Asp Asp Lys Gly Ala Pro Trp Ser His 1 5 10 15 Pro Gln Phe Glu Lys 20 31 24 PRT artificial sequence Description of Artificial Sequence synthetic module 31 Glu Phe Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn Gly Ala Pro 1 5 10 15 Trp Ser His Pro Gln Phe Glu Lys 20 32 17 PRT artificial sequence Description of Artificial Sequence synthetic module 32 Glu Phe Pro Gly Gly Ser Gly Gly Ala Pro His His His His His His 1 5 10 15 Cys 33 22 PRT artificial sequence Description of Artificial Sequence synthetic module 33 Cys Glu Phe Asp Tyr Lys Asp Asp Asp Asp Lys Gly Ala Pro Trp Ser 1 5 10 15 His Pro Gln Phe Glu Lys 20 34 25 PRT artificial sequence Description of Artificial Sequence synthetic module 34 Cys Glu Phe Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn Gly Ala 1 5 10 15 Pro Trp Ser His Pro Gln Phe Glu Lys 20 25 35 4380 DNA artificial sequence Description of Artificial Sequence vector 35 tctagagcat gcgtaggaga aaataaaatg aaacaaagca ctattgcact ggcactctta 60 ccgttgctct tcacccctgt taccaaagcc gactacaaag atgaagtgca attggtggaa 120 agcggcggcg gcctggtgca accgggcggc agcctgcgtc tgagctgcgc ggcctccgga 180 tttaccttta gcagctatgc gatgagctgg gtgcgccaag cccctgggaa gggtctcgag 240 tgggtgagcg cgattagcgg tagcggcggc agcacctatt atgcggatag cgtgaaaggc 300 cgttttacca tttcacgtga taattcgaaa aacaccctgt atctgcaaat gaacagcctg 360 cgtgcggaag atacggccgt gtattattgc gcgcgtcgtt ctggtgctta tgattattgg 420 ggccaaggca ccctggtgac ggttagctca gcgggtggcg gttctggcgg cggtgggagc 480 ggtggcggtg gttctggcgg tggtggttcc gatatcgtga tgacccagag cccggatagc 540 ctggcggtga gcctgggcga acgtgcgacc attaactgca gaagcagcca gagcgtgctg 600 tatagcagca acaacaaaaa ctatctggcg tggtaccagc agaaaccagg tcagccgccg 660 aaactattaa tttattgggc atccacccgt gaaagcgggg tcccggatcg ttttagcggc 720 tctggatccg gcactgattt taccctgacc atttcgtccc tgcaagctga agacgtggcg 780 gtgtattatt gccagcagta ttcttctttt cctcttacct ttggccaggg tacgaaagtt 840 gaaattaaac gtacggaatt cccagggggg agcggaggcg cgccgcacca tcatcaccat 900 cactgataag cttgacctgt gaagtgaaaa atggcgcaga ttgtgcgaca ttttttttgt 960 ctgccgttta attaaagggg ggggggggcc ggcctggggg ggggtgtaca tgaaattgta 1020 aacgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac 1080 caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg 1140 agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa 1200 gggcgaaaaa ccgtctatca gggcgatggc ccactacgag aaccatcacc ctaatcaagt 1260 tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt 1320 agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga 1380 gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc 1440 gcgcttaatg cgccgctaca gggcgcgtgc tagactagtg tttaaaccgg accggggggg 1500 ggcttaagtg ggctgcaaaa caaaacggcc tcctgtcagg aagccgcttt tatcgggtag 1560 cctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcatcag tgaatcggcc 1620 aacgcgcggg gagaggcggt ttgcgtattg ggagccaggg tggtttttct tttcaccagt 1680 gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg 1740 tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggtcag cggcgggata 1800 taacatgagc tgtcctcggt atcgtcgtat cccactaccg agatgtccgc accaacgcgc 1860 agcccggact cggtaatggc acgcattgcg cccagcgcca tctgatcgtt ggcaaccagc 1920 atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg 1980 gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta 2040 tgccagccag ccagacgcag acgcgccgag acagaactta atgggccagc taacagcgcg 2100 atttgctggt ggcccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcctcatgg 2160 gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca 2220 ttagtgcagg cagcttccac agcaatagca tcctggtcat ccagcggata gttaataatc 2280 agcccactga cacgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg 2340 cttcgttcta ccatcgacac gaccacgctg gcacccagtt gatcggcgcg agatttaatc 2400 gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc 2460 aacgactgtt tgcccgccag ttgttgtgcc acgcggttag gaatgtaatt cagctccgcc 2520 atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg 2580 cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt 2640 ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag 2700 gttttgcgcc attcgatgct agccatgtga gcaaaaggcc agcaaaaggc caggaaccgt 2760 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa 2820 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt 2880 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg 2940 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc 3000 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc 3060 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta 3120 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct 3180 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc 3240 tgcgctctgc tgtagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa 3300 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa 3360 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa 3420 aactcacgtt aagggatttt ggtcagatct agcaccaggc gtttaagggc accaataact 3480 gccttaaaaa aattacgccc cgccctgcca ctcatcgcag tactgttgta attcattaag 3540 cattctgccg acatggaagc catcacaaac ggcatgatga acctgaatcg ccagcggcat 3600 cagcaccttg tcgccttgcg tataatattt gcccatagtg aaaacggggg cgaagaagtt 3660 gtccatattg gctacgttta aatcaaaact ggtgaaactc acccagggat tggctgagac 3720 gaaaaacata ttctcaataa accctttagg gaaataggcc aggttttcac cgtaacacgc 3780 cacatcttgc gaatatatgt gtagaaactg ccggaaatcg tcgtggtatt cactccagag 3840 cgatgaaaac gtttcagttt gctcatggaa aacggtgtaa caagggtgaa cactatccca 3900 tatcaccagc tcaccgtctt tcattgccat acggaactcc gggtgagcat tcatcaggcg 3960 ggcaagaatg tgaataaagg ccggataaaa cttgtgctta tttttcttta cggtctttaa 4020 aaaggccgta atatccagct gaacggtctg gttataggta cattgagcaa ctgactgaaa 4080 tgcctcaaaa tgttctttac gatgccattg ggatatatca acggtggtat atccagtgat 4140 ttttttctcc attttagctt ccttagctcc tgaaaatctc gataactcaa aaaatacgcc 4200 cggtagtgat cttatttcat tatggtgaaa gttggaacct cacccgacgt ctaatgtgag 4260 ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg 4320 tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg attacgaatt 4380 36 2839 DNA artificial sequence Description of Artificial Sequence vector 36 acccgacacc atcgaaatta atacgactca ctatagggag accacaacgg tttcccgaat 60 tgtgagcgga taacaataga aataattttg tttaacttta agaaggagat atatccatgg 120 ctgaaactgt tgaaagttgt ttagcaaaat cccatacaga aaattcattt actaacgtct 180 ggaaagacga caaaacttta gatcgttacg ctaactatga gggctgtctg tggaatgcta 240 caggcgttgt agtttgtact ggtgacgaaa ctcagtgtta cggtacatgg gttcctattg 300 ggcttgctat ccctgaaaat gagggtggtg gctctgaggg tggcggttct ccgtacgacg 360 ttccagacta cgcttccctg cgttcccatc accatcacca tcactaagct tcagtcccgg 420 gcagtggatc cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct 480 gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg 540 aaaggaggaa ctatatccgg atcgagatcc ccacgcgccc tgtagcggcg cattaagcgc 600 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc 660 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct 720 aaatcggggc atccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa 780 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc 840 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact 900 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg 960 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt 1020 tacaatttca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt 1080 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata 1140 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt 1200 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc 1260 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat 1320 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct 1380 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca 1440 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg 1500 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa 1560 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg 1620 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga 1680 cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg 1740 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt 1800 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg 1860 agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc 1920 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca 1980 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc 2040 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat 2100 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc 2160 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg 2220 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct 2280 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct 2340 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct 2400 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg 2460 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc 2520 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga 2580 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg 2640 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta 2700 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg 2760 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg 2820 ctggcctttt gctcacatg 2839 37 4045 DNA artificial sequence Description of Artificial Sequence vector 37 agcttaatta gctgagcttg gactcctgtt gatagatcca gtaatgacct cagaactcca 60 tctggatttg ttcagaacgc tcggttgccg ccgggcgttt tttattggtg agaatccaag 120 ctagcttggc gagattttca ggagctaagg aagctaaaat ggagaaaaaa atcactggat 180 ataccaccgt tgatatatcc caatggcatc gtaaagaaca ttttgaggca tttcagtcag 240 ttgctcaatg tacctataac cagaccgttc agctggatat tacggccttt ttaaagaccg 300 taaagaaaaa taagcacaag ttttatccgg cctttattca cattcttgcc cgcctgatga 360 atgctcatcc ggaatttcgt atggcaatga aagacggtga gctggtgata tgggatagtg 420 ttcacccttg ttacaccgtt ttccatgagc aaactgaaac gttttcatcg ctctggagtg 480 aataccacga cgatttccgg cagtttctac acatatattc gcaagatgtg gcgtgttacg 540 gtgaaaacct ggcctatttc cctaaagggt ttattgagaa tatgtttttc gtctcagcca 600 atccctgggt gagtttcacc agttttgatt taaacgtggc caatatggac aacttcttcg 660 cccccgtttt caccatgcat gggcaaatat tatacgcaag gcgacaaggt gctgatgccg 720 ctggcgattc aggttcatca tgccgtctgt gatggcttcc atgtcggcag aatgcttaat 780 gaattacaac agtactgcga tgagtggcag ggcggggcgt aattttttta aggcagttat 840 tggtgccctt aaacgcctgg ggtaatgact ctctagcttg aggcatcaaa taaaacgaaa 900 ggctcagtcg aaagactggg cctttcgttt tatctgttgt ttgtcggtga acgctctcct 960 gagtaggaca aatccgccgc tctagagctg cctcgcgcgt ttcggtgatg acggtgaaaa 1020 cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag 1080 cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggcg cagccatgac 1140 ccagtcacgt agcgatagcg gagtgtatac tggcttaact atgcggcatc agagcagatt 1200 gtactgagag tgcaccatat gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac 1260 cgcatcaggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt ctgtcggctg 1320 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat 1380 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc 1440 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc 1500 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga 1560 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt 1620 ctcccttcgg gaagcgtggc gctttctcaa tgctcacgct gtaggtatct cagttcggtg 1680 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc 1740 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg 1800 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc 1860 ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg 1920 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc 1980 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct 2040 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt 2100 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa 2160 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa 2220 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagctgcc 2280 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct 2340 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca 2400 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt 2460 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt 2520 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc 2580 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc 2640 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt 2700 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact 2760 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc 2820 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt 2880 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg 2940 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct 3000 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa 3060 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt 3120 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc 3180 acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcat gacattaacc 3240 tataaaaata ggcgtatcac gaggcccttt cgtcttcacc tcgagaaatc ataaaaaatt 3300 tatttgcttt gtgagcggat aacaattata atagattcaa ttgtgagcgg ataacaattt 3360 cacacagaat tcattaaaga ggagaaatta accatgagtg acattgcctt cttgattgat 3420 ggctctggta gcatcatccc acatgacttt cggcggatga aggagtttgt ctcaactgtg 3480 atggagcaat taaaaaagtc caaaaccttg ttctctttga tgcagtactc tgaagaattc 3540 cggattcact ttaccttcaa agagttccag aacaacccta acccaagatc actggtgaag 3600 ccaataacgc agctgcttgg gcggacacac acggccacgg gcatccgcaa agtggtacga 3660 gagctgttta acatcaccaa cggagcccga aagaatgcct ttaagatcct agttgtcatc 3720 acggatggag aaaagtttgg cgatcccttg ggatatgagg atgtcatccc tgaggcagac 3780 agagagggag tcattcgcta cgtcattggg gtgggagatg ccttccgcag tgagaaatcc 3840 cgccaagagc ttaataccat cgcatccaag ccgcctcgtg atcacgtgtt ccaggtgaat 3900 aactttgagg ctctgaagac cattcagaac cagcttcggg agaagatctt tgcgatcgag 3960 ggtactcaga caggaagtag cagctccttt gagcatgaga tgtctcagga aatcgaaggt 4020 agacatcacc atcaccatca ctaga 4045 38 1574 DNA artificial sequence Description of Artificial Sequence expression cassette 38 gctagcctga ggccagtttg ctcaggctct ccccgtggag gtaataattg ctcgaccgat 60 aaaagcggct tcctgacagg aggccgtttt gttttgcagc ccacctcaac gcaattaatg 120 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt 180 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg 240 aatttctaga taacgagggc aaaaaatgaa aaagacagct atcgcgattg cagtggcact 300 ggctggtttc gctaccgtag cgcaggccga ctactgcgat atcgaattcg cagaaacagt 360 tgaaagttgt ttagcaaaac cccatacaga aaattcattt actaacgtct ggaaagacga 420 caaaacttta gatcgttacg ctaactatga gggctgtctg tggaatgcta caggcgttgt 480 agtttgtact ggtgacgaaa ctcagtgtta cggtacatgg gttcctattg ggcttgctat 540 ccctgaaaat gagggtggtg gctctgaggg tggcggttct gagggtggcg gctctgaggg 600 tggcggtact aaacctcctg agtacggtga tacacctatt ccgggctata cttatatcaa 660 ccctctcgac ggcacttatc cgcctggtac tgagcaaaac cccgctaatc ctaatccttc 720 tcttgaggag tctcagcctc ttaatacttt catgtttcag aataataggt tccgaaatag 780 gcagggggca ttaactgttt atacgggcac tgttactcaa ggcactgacc ccgttaaaac 840 ttattaccag tacactcctg tatcatcaaa agccatgtat gacgcttact ggaacggtaa 900 attcagagac tgcgctttcc attctggctt taatgaggat ccattcgttt gtgaatatca 960 aggccaatcg tctgacctgc ctcaacctcc tgtcaatgct ggcggcggct ctggtggtgg 1020 ttctggtggc ggctctgagg gtggcggctc tgagggtggc ggttctgagg gtggcggctc 1080 tgagggtggc ggttccggtg gcggctccgg ttccggtgat tttgattatg aaaaaatggc 1140 aaacgctaat aagggggcta tgaccgaaaa tgccgatgaa aacgcgctac agtctgacgc 1200 taaaggcaaa cttgattctg tcgctactga ttacggtgct gctatcgatg gtttcattgg 1260 tgacgtttcc ggccttgcta atggtaatgg tgctactggt gattttgctg gctctaattc 1320 ccaaatggct caagtcggtg acggtgataa ttcaccttta atgaataatt tccgtcaata 1380 tttaccttct ttgcctcagt cggttgaatg tcgcccttat gtctttggcg ctggtaaacc 1440 atatgaattt tctattgatt gtgacaaaat aaacttattc cgtggtgtct ttgcgtttct 1500 tttatatgtt gccaccttta tgtatgtatt ttcgacgttt gctaacatac tgcgtaataa 1560 ggagtcttaa gctt 1574 39 932 DNA artificial sequence Description of Artificial Sequence expression cassette 39 gctagcctga ggccagtttg ctcaggctct ccccgtggag gtaataattg ctcgaccgat 60 aaaagcggct tcctgacagg aggccgtttt gttttgcagc ccacctcaac gcaattaatg 120 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt 180 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg 240 aatttctaga taacgagggc aaaaaatgaa aaagacagct atcgcgattg cagtggcact 300 ggctggtttc gctaccgtag cgcaggccga ctactgcgat atcgaattca atgctggcgg 360 cggctctggt ggtggttctg gtggcggctc tgagggtggt ggctctgagg gtggcggttc 420 tgagggtggc ggctctgagg gaggcggttc cggtggtggc tctggttccg gtgattttga 480 ttatgaaaag atggcaaacg ctaataaggg ggctatgacc gaaaatgccg atgaaaacgc 540 gctacagtct gacgctaaag gcaaacttga ttctgtcgct actgattacg gtgctgctat 600 cgatggtttc attggtgacg tttccggcct tgctaatggt aatggtgcta ctggtgattt 660 tgctggctct aattcccaaa tggctcaagt cggtgacggt gataattcac ctttaatgaa 720 taatttccgt caatatttac cttccctccc tcaatcggtt gaatgtcgcc cttttgtctt 780 tggcgctggt aaaccatatg aattttctat tgattgtgac aaaataaact tattccgtgg 840 tgtctttgcg tttcttttat atgttgccac ctttatgtat gtattttcta cgtttgctaa 900 catactgcgt aataaggagt cttgataagc tt 932 40 4425 DNA artificial sequence Description of Artificial Sequence vector 40 tctagataac gagggcaaaa aatgaaaaag acagctatcg cgattgcagt ggcactggct 60 ggtttcgcta ccgtagcgca ggccgactac tgcgatatcg aattcgcaga aacagttgaa 120 agttgtttag caaaacccca tacagaaaat tcatttacta acgtctggaa agacgacaaa 180 actttagatc gttacgctaa ctatgagggc tgtctgtgga atgctacagg cgttgtagtt 240 tgtactggtg acgaaactca gtgttacggt acatgggttc ctattgggct tgctatccct 300 gaaaatgagg gtggtggctc tgagggtggc ggttctgagg gtggcggctc tgagggtggc 360 ggtactaaac ctcctgagta cggtgataca cctattccgg gctatactta tatcaaccct 420 ctcgacggca cttatccgcc tggtactgag caaaaccccg ctaatcctaa tccttctctt 480 gaggagtctc agcctcttaa tactttcatg tttcagaata ataggttccg aaataggcag 540 ggggcattaa ctgtttatac gggcactgtt actcaaggca ctgaccccgt taaaacttat 600 taccagtaca ctcctgtatc atcaaaagcc atgtatgacg cttactggaa cggtaaattc 660 agagactgcg ctttccattc tggctttaat gaggatccat tcgtttgtga atatcaaggc 720 caatcgtctg acctgcctca acctcctgtc aatgctggcg gcggctctgg tggtggttct 780 ggtggcggct ctgagggtgg cggctctgag ggtggcggtt ctgagggtgg cggctctgag 840 ggtggcggtt ccggtggcgg ctccggttcc ggtgattttg attatgaaaa aatggcaaac 900 gctaataagg gggctatgac cgaaaatgcc gatgaaaacg cgctacagtc tgacgctaaa 960 ggcaaacttg attctgtcgc tactgattac ggtgctgcta tcgatggttt cattggtgac 1020 gtttccggcc ttgctaatgg taatggtgct actggtgatt ttgctggctc taattcccaa 1080 atggctcaag tcggtgacgg tgataattca cctttaatga ataatttccg tcaatattta 1140 ccttctttgc ctcagtcggt tgaatgtcgc ccttatgtct ttggcgctgg taaaccatat 1200 gaattttcta ttgattgtga caaaataaac ttattccgtg gtgtctttgc gtttctttta 1260 tatgttgcca cctttatgta tgtattttcg acgtttgcta acatactgcg taataaggag 1320 tcttaaggcc tgataagcat gcgtaggaga aaataaaatg aaacaaagca ctattgcact 1380 ggcactctta ccgttgctct tcacccctgt taccaaagcc gactacaaag atgaagtgca 1440 attggtggaa agcggcggcg gcctggtgca accgggcggc agcctgcgtc tgagctgcgc 1500 ggcctccgga tttaccttta gcagctatgc gatgagctgg gtgcgccaag cccctgggaa 1560 gggtctcgag tgggtgagcg cgattagcgg tagcggcggc agcacctatt atgcggatag 1620 cgtgaaaggc cgttttacca tttcacgtga taattcgaaa aacaccctgt atctgcaaat 1680 gaacagcctg cgtgcggaag atacggccgt gtattattgc gcgcgtcgtt ctggtgctta 1740 tgattattgg ggccaaggca ccctggtgac ggttagctca gcgggtggcg gttctggcgg 1800 cggtgggagc ggtggcggtg gttctggcgg tggtggttcc gatatcgtga tgacccagag 1860 cccggatagc ctggcggtga gcctgggcga acgtgcgacc attaactgca gaagcagcca 1920 gagcgtgctg tatagcagca acaacaaaaa ctatctggcg tggtaccagc agaaaccagg 1980 tcagccgccg aaactattaa tttattgggc atccacccgt gaaagcgggg tcccggatcg 2040 ttttagcggc tctggatccg gcactgattt taccctgacc atttcgtccc tgcaagctga 2100 agacgtggcg gtgtattatt gccagcagta ttcttctttt cctcttacct ttggccaggg 2160 tacgaaagtt gaaattaaac gtacggaatt cccagggggg agcggaggcg cgccgcacca 2220 tcatcaccat cactgctgat aagcttgacc tgtgaagtga aaaatggcgc agattgtgcg 2280 acattttttt tgtctgccgt ttaatgaaat tgtaaacgtt aatattttgt taaaattcgc 2340 gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc 2400 ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag 2460 tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga 2520 tggcccacta cgagaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc 2580 actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa 2640 cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt 2700 agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc 2760 gtgctagcca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg 2820 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt 2880 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc 2940 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct 3000 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc 3060 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agtccgaccg ctgcgcctta 3120 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca 3180 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag 3240 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgtag 3300 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt 3360 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa 3420 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg 3480 attttggtca gatctagcac caggcgttta agggcaccaa taactgcctt aaaaaaatta 3540 cgccccgccc tgccactcat cgcagtactg ttgtaattca ttaagcattc tgccgacatg 3600 gaagccatca caaacggcat gatgaacctg aatcgccagc ggcatcagca ccttgtcgcc 3660 ttgcgtataa tatttgccca tagtgaaaac gggggcgaag aagttgtcca tattggctac 3720 gtttaaatca aaactggtga aactcaccca gggattggct gagacgaaaa acatattctc 3780 aataaaccct ttagggaaat aggccaggtt ttcaccgtaa cacgccacat cttgcgaata 3840 tatgtgtaga aactgccgga aatcgtcgtg gtattcactc cagagcgatg aaaacgtttc 3900 agtttgctca tggaaaacgg tgtaacaagg gtgaacacta tcccatatca ccagctcacc 3960 gtctttcatt gccatacgga actccgggtg agcattcatc aggcgggcaa gaatgtgaat 4020 aaaggccgga taaaacttgt gcttattttt ctttacggtc tttaaaaagg ccgtaatatc 4080 cagctgaacg gtctggttat aggtacattg agcaactgac tgaaatgcct caaaatgttc 4140 tttacgatgc cattgggata tatcaacggt ggtatatcca gtgatttttt tctccatttt 4200 agcttcctta gctcctgaaa atctcgataa ctcaaaaaat acgcccggta gtgatcttat 4260 ttcattatgg tgaaagttgg aacctcaccc gacgtctaat gtgagttagc tcactcatta 4320 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg 4380 ataacaattt cacacaggaa acagctatga ccatgattac gaatt 4425 41 5079 DNA artificial sequence Description of Artificial Sequence vector 41 tctagataac gagggcaaaa aatgaaaaag acagctatcg cgattgcagt ggcactggct 60 ggtttcgcta ccgtagcgca ggccgatatc gtgctgaccc agccgccttc agtgagtggc 120 gcaccaggtc agcgtgtgac catctcgtgt agcggcagca gcagcaacat tggcagcaac 180 tatgtgagct ggtaccagca gttgcccggg acggcgccga aactgctgat ttatgataac 240 aaccagcgtc cctcaggcgt gccggatcgt tttagcggat ccaaaagcgg caccagcgcg 300 agccttgcga ttacgggcct gcaaagcgaa gacgaagcgg attattattg ccagagctat 360 gaccagaatg ctcttgttga ggtgtttggc ggcggcacga agttaaccgt tcttggccag 420 ccgaaagccg caccgagtgt gacgctgttt ccgccgagca gcgaagaatt gcaggcgaac 480 aaagcgaccc tggtgtgcct gattagcgac ttttatccgg gagccgtgac agtggcctgg 540 aaggcagata gcagccccgt caaggcggga gtggagacca ccacaccctc caaacaaagc 600 aacaacaagt acgcggccag cagctatctg agcctgacgc ctgagcagtg gaagtcccac 660 agaagctaca gctgccaggt cacgcatgag gggagcaccg tggaaaaaac cgttgcgccg 720 actgaggcct ctccaggggg gagcggaggc gcgccgcacc atcatcacca tcactgctga 780 taatatgcat gcgtaggaga aaataaaatg aaacaaagca ctattgcact ggcactctta 840 ccgttgctct tcacccctgt taccaaagcc caggtgcaat tgaaagaaag cggcccggcc 900 ctggtgaaac cgacccaaac cctgaccctg acctgtacct tttccggatt tagcctgtcc 960 acgtctggcg ttggcgtggg ctggattcgc cagccgcctg ggaaagccct cgagtggctg 1020 gctctgattg attgggatga tgataagtat tatagcacca gcctgaaaac gcgtctgacc 1080 attagcaaag atacttcgaa aaatcaggtg gtgctgacta tgaccaacat ggacccggtg 1140 gatacggcca cctattattg cgcgcgtttt gatccttttt ttgattcttt ttttgattat 1200 tggggccaag gcaccctggt gacggttagc tcagcgtcga ccaaaggtcc aagcgtgttt 1260 ccgctggctc cgagcagcaa aagcaccagc ggcggcacgg ctgccctggg ctgcctggtt 1320 aaagattatt tcccggaacc agtcaccgtg agctggaaca gcggggcgct gaccagcggc 1380 gtgcatacct ttccggcggt gctgcaaagc agcggcctgt atagcctgag cagcgttgtg 1440 accgtgccga gcagcagctt aggcactcag acctatattt gcaacgtgaa ccataaaccg 1500 agcaacacca aagtggataa aaaagtggaa ccgaaaagcg aattcgacta taaagatgac 1560 gatgacaaag gcgcgccgtg gagccacccg cagtttgaaa aatgataagc ttgacctgtg 1620 aagtgaaaaa tggcgcagat tgtgcgacat tttttttgtc tgccgtttaa ttaaaggggg 1680 gggggggccg gcctgggggg gggtgtacat gaaattgtaa acgttaatat tttgttaaaa 1740 ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa 1800 atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac 1860 aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag 1920 ggcgatggcc cactacgaga accatcaccc taatcaagtt ttttggggtc gaggtgccgt 1980 aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg 2040 gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca 2100 agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag 2160 ggcgcgtgct agactagtgt ttaaaccgga ccgggggggg gcttaagtgg gctgcaaaac 2220 aaaacggcct cctgtcagga agccgctttt atcgggtagc ctcactgccc gctttccagt 2280 cgggaaacct gtcgtgccag ctgcatcagt gaatcggcca acgcgcgggg agaggcggtt 2340 tgcgtattgg gagccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg 2400 cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc 2460 aggcgaaaat cctgtttgat ggtggtcagc ggcgggatat aacatgagct gtcctcggta 2520 tcgtcgtatc ccactaccga gatgtccgca ccaacgcgca gcccggactc ggtaatggca 2580 cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc 2640 tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt 2700 tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga 2760 cgcgccgaga cagaacttaa tgggccagct aacagcgcga tttgctggtg gcccaatgcg 2820 accagatgct ccacgcccag tcgcgtaccg tcctcatggg agaaaataat actgttgatg 2880 ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca 2940 gcaatagcat cctggtcatc cagcggatag ttaataatca gcccactgac acgttgcgcg 3000 agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacg 3060 accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc 3120 gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt 3180 tgttgtgcca cgcggttagg aatgtaattc agctccgcca tcgccgcttc cactttttcc 3240 cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag 3300 acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat 3360 tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatgcta 3420 gccatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg 3480 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg 3540 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg 3600 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga 3660 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc 3720 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt 3780 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact 3840 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg 3900 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gtagccagtt 3960 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt 4020 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct 4080 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg 4140 gtcagatcta gcaccaggcg tttaagggca ccaataactg ccttaaaaaa attacgcccc 4200 gccctgccac tcatcgcagt actgttgtaa ttcattaagc attctgccga catggaagcc 4260 atcacaaacg gcatgatgaa cctgaatcgc cagcggcatc agcaccttgt cgccttgcgt 4320 ataatatttg cccatagtga aaacgggggc gaagaagttg tccatattgg ctacgtttaa 4380 atcaaaactg gtgaaactca cccagggatt ggctgagacg aaaaacatat tctcaataaa 4440 ccctttaggg aaataggcca ggttttcacc gtaacacgcc acatcttgcg aatatatgtg 4500 tagaaactgc cggaaatcgt cgtggtattc actccagagc gatgaaaacg tttcagtttg 4560 ctcatggaaa acggtgtaac aagggtgaac actatcccat atcaccagct caccgtcttt 4620 cattgccata cggaactccg ggtgagcatt catcaggcgg gcaagaatgt gaataaaggc 4680 cggataaaac ttgtgcttat ttttctttac ggtctttaaa aaggccgtaa tatccagctg 4740 aacggtctgg ttataggtac attgagcaac tgactgaaat gcctcaaaat gttctttacg 4800 atgccattgg gatatatcaa cggtggtata tccagtgatt tttttctcca ttttagcttc 4860 cttagctcct gaaaatctcg ataactcaaa aaatacgccc ggtagtgatc ttatttcatt 4920 atggtgaaag ttggaacctc acccgacgtc taatgtgagt tagctcactc attaggcacc 4980 ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca 5040 atttcacaca ggaaacagct atgaccatga ttacgaatt 5079 

1. A method for displaying a (poly)peptide/protein on the surface of a bacteriophage particle comprising: causing or allowing the attachment of said (poly)peptide/protein after expression to a member of the protein coat of said bacteriophage particle, wherein said attachment is caused by the formation of a disulfide bond between a first cysteine residue comprised in said (poly)peptide/protein and a second cysteine residue comprised in said member of the protein coat.
 2. The method of claim 1, wherein said second cysteine residue is present at a corresponding amino acid position in a wild type coat protein of a bacteriophage.
 3. The method of claim 2, wherein said member of the protein coat is a wild type coat protein of a bacteriophage.
 4. The method of claim 2, wherein said member of the protein coat is a truncated variant of a wild type coat protein of a bacteriophage, wherein said truncated variant comprises at least that part of said wild type coat protein causing the incorporation of said coat protein into the protein coat of the bacteriophage particle.
 5. The method of claim 2, wherein said member of the protein coat is a modified variant of a wild type coat protein of a bacteriophage, wherein said modified variant is capable of being incorporated into the protein coat of the bacteriophage particle.
 6. The method of claim 1, wherein said second cysteine residue is not present at a corresponding amino acid position in a wild type coat protein of a bacteriophage.
 7. The method of claim 6, wherein said second cysteine has been artificially introduced into a wild type coat protein of a bacteriophage.
 8. The method of claim 6, wherein said second cysteine has been artificially introduced into a truncated variant of a wild type coat protein of a bacteriophage.
 9. The method of claim 6, wherein said second cysteine has been artificially introduced into a modified variant of a wild type coat protein of a bacteriophage.
 10. The method of any one of claims 4 to 9, wherein said second cysteine is present at, or in the vicinity of, the C-or the N-terminus of said member of the phage coat of said bacteriophage particle.
 11. The method of any one of claims 1 to 10, wherein said bacteriophage is a filamentous bacteriophage.
 12. The method of claim 11, wherein said member of the protein coat of the bacteriophage particle is or is derived from the wild type coat protein pIII.
 13. The method of claim 11, wherein said member of the protein coat of the bacteriophage particle is or is derived from the wild type coat protein pIX.
 14. The method of any one of claims 1 to 12, comprising: (a) providing a host cell harbouring a nucleic acid sequence comprising a nucleic acid sequence encoding said (poly)peptide/protein; (b) causing or allowing the expression of said nucleic acid sequence; and (c) causing or allowing the production of bacteriophage particles in said host cell.
 15. The method of any one of claims 1 to 14, wherein said (poly)peptide/protein comprises an immunoglobulin or a functional fragment thereof.
 16. The method of claim 15, wherein said functional fragment is an scFv or Fab fragment.
 17. A nucleic acid sequence encoding a modified variant of a wild type coat protein of a bacteriophage, wherein said modified variant consists of: (a) one or more parts of said wild type coat protein of a bacteriophage, wherein one of said parts comprises at least that part which causes or allows the incorporation of said coat protein into the phage coat; and (b) between one and six additional amino acid residues not present at the corresponding amino acid positions in a wild type coat protein of a bacteriophage, wherein one of said additional amino acid residues is a cysteine residue.
 18. A nucleic acid sequence encoding a modified variant of a wild type coat protein of a bacteriophage, wherein said modified variant consists of (a) one or more parts of said wild type coat protein of a bacteriophage, wherein one of said parts comprises at least that part which causes or allows the incorporation of said coat protein into the phage coat; (b) between one and six additional amino acid residues not present at the corresponding amino acid positions in a wild type coat protein of a bacteriophage, wherein one of said additional amino acid residues is a cysteine residue; and (c) one or more peptide sequences for purification and/or detection purposes.
 19. A vector comprising the nucleic acid of claim 17 or
 18. 20. The vector of claim 19, further comprising one or more nucleic acid sequences encoding a (poly)peptide/protein comprising a second cysteine residue.
 21. The vector of claim 20, wherein said (poly)peptide/protein comprises an immunoglobulin or a functional fragment thereof.
 22. A host cell comprising the nucleic acid sequence of claim 17 or 18, the vector of any one of claims 19 to
 21. 23. A modified variant of a wild type coat protein of a bacteriophage encoded by the nucleic acid sequence of claim 17 or 18, the vector of any one of claims 19 to 21, or produced by the host cell of claim
 22. 24. A bacteriophage particle displaying a (poly)peptide/protein on its surface obtainable by a method comprising: causing or allowing the attachment of said (poly)peptide/protein after expression to a member of the protein coat of said bacteriophage particle, wherein said attachment is caused by the formation of a disulfide bond between a first cysteine residue comprised in said (poly)peptide/protein and a second cysteine residue comprised in said member of the protein coat.
 25. A bacteriophage particle displaying a (poly)peptide/protein attached to its surface, wherein said attachment is caused by the formation of a disulfide bond between a first cysteine residue comprised in said (poly)peptide/protein and a second cysteine residue comprised in a member of the protein coat of said bacteriophage particle.
 26. The bacteriophage particle of claim 24 or 25, further comprising a vector comprising one or more nucleic acid sequences encoding said (poly)peptide/protein.
 27. The bacteriophage particle of claim 26, wherein said vector is the vector of claim 20 or
 21. 28. A diverse collection of bacteriophage particles of any one of claims 25 to 27, wherein each of said bacteriophage particles displays a (poly)peptide/protein out of a diverse collection of (poly)peptides/proteins.
 29. A method for obtaining a (poly)peptide/protein having a desired property, comprising: (a) providing the diverse collection of bacteriophage particles of claim 28; and (b) screening said diverse collection and/or selecting from said diverse collection to obtain at least one bacteriophage particle displaying a (poly)peptide/protein having said desired property.
 30. The method of claim 29, wherein said desired property is binding to a target of interest.
 31. The method of claim 30, wherein step (b) further comprises: (ba) contacting said diverse collection of bacteriophage particles with the target of interest; (bb) eluting bacteriophage particles not binding to the target of interest; (bc) eluting bacteriophage particles binding to the target of interest by treating the complexes of target of interest and bacteriophages binding to said target of interest formed in step (ba) under reducing conditions. 